Antibacterial Activity of an Ethyl Acetate Extract of <italic>Pseudomonas</italic> sp. UJ-6 against Methicillin-Resistant <italic>Staphylococcus aureus</italic>
Antibacterial Activity of an Ethyl Acetate Extract of Pseudomonas sp. UJ-6 against Methicillin-Resistant Staphylococcus aureus
Fisheries and aquatic sciences. 2013. Jun, 16(2): 79-84
Copyright ©2013, The Korean Society of Fisheries and Aquatic Science
This is an Open Access article distributed under the terms of the CreativeCommons Attribution Non-Commercial License ( which permits unrestricted non-commercial use,distribution, and reproduction in any medium, provided the original workis properly cited.
  • Received : May 15, 2013
  • Accepted : June 03, 2013
  • Published : June 30, 2013
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About the Authors
Dae-Sung Lee
POSTECH Ocean Science and Technology Institute, POSTECH, Pohang 790-784, Korea
Sung-Hwan Eom
Department of Food Science and Technology, Pukyong National University, Busan 608-737, Korea
Jae-Young Je
Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu 550-749, Korea
Young-Mog Kim
Department of Food Science and Technology, Pukyong National University, Busan 608-737, Korea
Myung-Suk Lee
Department of Microbiology, Pukyong National University, Busan 608-737, Korea
Young-Man Kim
Department of Food and Nutrition, Dong-Eui University, Busan 614-714, Korea

In an effort to discover an alternative antibiotic for treating infections with methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas sp. UJ-6, a marine bacterium that exhibited antibacterial activity against MRSA, was isolated. The culture broth and its ethyl acetate extract exhibited bactericidal activity against MRSA. The extract also exhibited antibacterial activity against gram-negative bacteria, which were not susceptible to vancomycin. The treatment of MRSA with the extract resulted in abnormal cell lysis. The extract retained >95% of its anti-MRSA activity after heat treatment for 15 min at 121℃. Thus, although most antibiotics are unstable under conditions of thermal stress, Pseudomonas sp. UJ-6 produces a heat-stable anti-MRSA substance. The results of this study strongly suggest that Pseudomonas sp. UJ-6 can be used to develop a novel, heat-stable, broad-spectrum antibiotic for the treatment of MRSA infections.
The emergence and increasing spread of antibiotic-resistant microorganisms, including nosocomial and community-acquired infections with Staphylococcus aureus , has become a serious public health problem (Schaberg et al., 1991; Witte, 1999; Levy, 2005). Since it was first detected in 1961 (Jevons, 1961), methicillin-resistant S. aureus (MRSA) has been considered to be a serious pathogen due to its resistance to almost all commercial antibiotics, high morbidity rate, and high mortality rate. Until recently, glycopeptide antibiotics such as vancomycin and teicoplanin were used as the last resort for the treatment of MRSA infections; however, glycopeptideresistant strains have emerged in several countries (Eom et al., 2012). Currently, several antibiotics, including linezolid, daptomycin, tigecycline, and quinupristin/dalfopristin, have been shown to possess anti-MRSA activity, but strains that are resistant to these antibiotics have been reported (Woodford, 2005). In view of these problems, the development of new anti-MRSA agents is urgently needed and many researchers have searched for alternative antibiotics against MRSA infections (Hiramatsu et al., 1997; Hanaki et al., 1998; Witte, 1999; Micek, 2007; Lee et al., 2008b; Eom et al., 2011).
Microorganisms are a source of antibacterial compounds; however, most are derived from terrestrial actinomycetes. Marine microbial metabolites provide the opportunity to produce novel antibiotics with unique chemical features, as compared to terrestrial ones, because marine microorganisms can live under harsh conditions such as high pressures, low food availability, total darkness, and extreme cold (Rahman et al., 2010; Abad et al., 2011). Indeed, several antibacterial compounds from marine microorganisms such as thiomarinols from Alteromonas rava (Shiozawa et al., 1997), bogorol A and loloatins from Bacillus sp. (Gerard et al., 1999; Barsby et al., 2001), agrochelin and sesbanimides from Agrobacterium (Acebal et al., 1998, 1999), pelagiomicins from Pelagiobacter variabilis (Imamura et al., 1997), δ-indomycinone and marinopyrroles from a Streptomyces sp. (Biabani et al., 1997; Hughes et al., 2008), MC21-A and -B from Pseudoalteromonas phenolica (Isnansetyo and Kamei, 2003, 2009), abyssomicin from Verrucosispora sp. (Keller et al., 2007), 2,4-diacetylphloroglucinol and 1-acetyl-beta-carboline from Pseudomonas sp. (Kamei and Isnansetyo, 2003; Lee et al., 2013), marinomycins and lynamicins from Marinispora sp. (Kwon et al., 2006; McArthur et al., 2008), kocurin from Kocuria palustris (Martín et al., 2013), dihydrophencomycin methyl ester from a streptomycete (Pusecker et al., 1997), and lipoxazolidinones from an actinomycete (Macherla et al., 2007), have been reported. Therefore, marine microorganisms have attracted great attention as potential sources of novel and effective compounds with antibacterial activity. The present study was conducted to investigate the antibacterial activity of an ethyl acetate extract against MRSA.
Materials and Methods
- Microorganisms and media
Pseudomonas sp. UJ-6 (GenBank accession no. GQ988399) exhibiting antibacterial activity against MRSA was isolated from seawater and incubated at 25℃ in PPES-II medium (0.2% polypeptone, 0.1% proteose peptone, 0.1% yeast extract, 0.1% soytone, and 0.001% ferric citrate, initial pH 7.6) The bacterial strains tested for antibacterial activity were purchased from the Korean Culture Center of Microorganisms (KCCM; Seoul, Korea) or the Korean Collection for Type Cultures (Daejeon, Korea); 13 clinical isolates of MRSA were provided by Donga-A University Hospital (Busan, Korea). The pathogenic bacteria were cultivated at 37℃ in Mueller-Hinton broth (Difco Laboratories, Detroit, MI, USA) for minimum inhibitory concentration (MIC) testing and on Mueller-Hinton agar plates (Difco Laboratories) for disk diffusion assays.
- Optimum culture conditions for Pseudomonas sp. UJ-6
The optimal temperature, pH, and NaCl concentration were determined for the culture of Pseudomonas sp. UJ-6 in PPES-II medium. To determine the optimal temperature, cells were incubated aerobically in PPES-II broth medium (pH 7, 2% NaCl) at different temperatures (4, 15, 20, 25, 30, 37, and 50℃). The pH range for growth was determined by incubating cells in PPES-II broth medium (2% NaCl, 25℃) at pH values ranging from 4-10. The salt tolerance of the cells was tested on PPES-II broth medium (pH 7, 25℃) supplemented with 0-10% NaCl (w/v).
- Relationship between Pseudomonas sp. UJ-6 cell growth and anti-MRSA activity
Broth from a Pseudomonas sp. UJ-6 culture grown at 25℃ in PPES-II medium was concentrated using a rotary vacuum evaporator and then mixed with Muller-Hinton broth containing MRSA strain KCCM 40510 at an estimated cell density of 10 4 CFU/mL. The cell growth of UJ-6 was monitored using the turbidity method at 640 nm. The anti-MRSA activity in the tube was evaluated based on viable cell counts of the MRSA strain after 24 h of incubation.
- Crude isolation of the anti-MRSA substance from a Pseudomonas sp. UJ-6 culture
Isolated UJ-6 was cultured in PPES-II broth medium at 25℃ with shaking at 150 rpm for 48 h, after which the cellfree supernatant was obtained by centrifugation (15,000 g at 4℃) and filtration (0.2-μm pore size membrane filter). The cell-free supernatant was partitioned by extraction with several organic solvents at a 1:1 (v/v) ratio according to their polarity, and the crude extracts were then concentrated using a rotary evaporator. The anti-MRSA activity of each fraction was tested, and the active fraction ( i.e. , the ethyl acetate fraction) was used as a crude antibiotic for further study.
- Measurement of the MIC
The two-fold serial dilution method was used to determine the MIC of the extract as described by the National Committee for Clinical Laboratory Standards (2004). The MIC of the crude extract was defined as the lowest concentration without growth after incubation at 37℃ for 24 h.
- Effect of the crude extract on MRSA cell morphology
To compare the effects of the crude extract on MRSA cell morphology, MRSA cells were incubated at 37℃ for 24 h in the presence or absence of the extract and then observed using a transmission electron microscope (JEM 1200EX-II; JEOL, Tokyo, Japan) at Pusan Paik Hospital (Busan, Korea).
- General characteristics of the crude extract
To investigate the thermal stability of the crude extract, the extract was incubated at several temperatures (4, 25, 50, 75, and 100℃) for 1 h. It was also autoclaved at 121℃ for 15
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Isolation of an bacterium exhibiting antibacterial activity against methicillin-resistant Staphylococcus aureus. Arrow indicated the isolated strain UJ-6.
min. To determine its pH stability, the crude extract was suspended in 0.1 M citrate phosphate buffer at a pH of 3 to 7 or 0.1 M Tris-HCl buffer at a pH of 8 to 10 for 30 min. After treatment, the anti-MRSA activity of the extract was estimated by the disk diffusion method.
Results and Discussion
- Culture characteristics of Pseudomonas sp. UJ-6
The anti-MRSA activity of Pseudomonas sp. UJ-6 is shown in Fig. 1 . To determine the optimal culture conditions for Pseudomonas sp. UJ-6, cells were incubated at different temperatures, pH values, and NaCl concentrations. Pseudomonas sp. UJ-6 was able to grow at temperatures ranging from 4 to 40℃, but not above 50℃. Also, the strain grew well between pH values of 5.0 and 9.0, but its growth was inhibited below pH 4.0 and above pH 10.0. A high concentration of NaCl (>4%) resulted in growth retardation or no growth (>8% NaCl). Thus, the most favorable growth of Pseudomonas sp. UJ-6 was observed in medium containing 1% NaCl, adjusted to pH 7.0, and incubated at 25℃ ( Fig. 2 ). However, there was no significant difference in anti-MRSA activity between different culture conditions (data not shown).
- Anti-MRSA activity of Pseudomonas sp. UJ-6
The supernatant of cultured Pseudomonas sp. UJ-6 showed bactericidal activity against MRSA, indicating that the strain produces an anti-bacterial substance. The strongest activity was observed after the stationary phase of growth ( Fig. 3 ). To elucidate the mechanism underlying the observed anti-MRSA activity and to purify the active compound from strain UJ-6,
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Effects of temperature (A), pH (B), and NaCl concentration (C) on the growth of Pseudomonas sp. UJ-6 in PPES-II medium.
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elationship between cell growth of Pseudomonas sp. UJ-6 and anti-MRSA activity. ○, Pseudomonas sp. UJ-6; ■, MRSA, methicillin-resistant Staphylococcus aureus.
a culture was extracted with several organic solvents, including ether, hexane, chloroform, methylene chloride, and ethyl acetate. Among these, only the ethyl acetate extract showed significant antibacterial activity against all of the tested grampositive species, including MRSA strains, and all tested gram-negative species. The MICs of the ethyl acetate extract against the MRSA strains and other bacteria are shown in Table 1 . The ethyl acetate extract showed antibacterial activity against the tested MRSA strains with MIC values ranging from 160 to 320 μg/mL. The extract also exhibited antibacterial activity against gram-negative bacteria, although it was less effective against gram-negative bacteria and Streptococcus iniae than against other Gram-positive bacteria. However, vancomycin was not effective against gram-negative bacteria (Totsuka et al., 1999; Lee et al., 2008b), suggesting that the anti-MRSA effect of the substance produced by UJ-6 differs from that of vancomycin. These results are similar to those reported for other marine bacteria producing an anti-MRSA substance (Isnansetyo and Kamei, 2003).
Antibacterial activity of the ethyl acetate extract of Pseudomonas sp. UJ-6 culturePseudomonassp. UJ-6 was cultured and extracted as described in Material and Methods.MRSA, methicillin-resistantStaphylococcus aureus, NA, not active.*Minimum inhibitory concentration (MIC) of crude extract and vancomycin was determined by the two-fold serial dilution method in Mueller- Hinton broth.
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Antibacterial activity of the ethyl acetate extract of Pseudomonas sp. UJ-6 culture Pseudomonas sp. UJ-6 was cultured and extracted as described in Material and Methods. MRSA, methicillin-resistant Staphylococcus aureus, NA, not active. *Minimum inhibitory concentration (MIC) of crude extract and vancomycin was determined by the two-fold serial dilution method in Mueller- Hinton broth.
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Abnormal cell morphology of methicillin-resistant Staphylococcus aureus (MRSA) caused by the ethyl acetate extract of Pseudomonas sp. UJ-6 culture. A MRSA strain (KCCM 40510, 105 CFU/mL) was inoculated in a Mueller-Hinton broth in the absence or presence of the ethyl acetate extract (320 μg/mL). The culture was incubated at 37℃ for 24 h and the cell morphology was observed with a transmission electron microscopy. (A) Normal cell of the MRSA (B) abnormal cell lysis of the MRSA grown with the ethyl acetate extract. Scale bars: A, B = 100 nm.
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The pH (A) and thermal stability (B) of the ethyl acetate extract of Pseudomonas sp. UJ-6 culture. For pH stability, the extract was suspended in 0.1 M citrate phosphate buffer for the range of pH 3 to 7 and 0.1 M Tris- HCl buffer for pH 8 to 10, and then kept in each buffer for 30 min. For thermal stability, the extract was incubated at an indicated temperature (4, 25, 50, 75, and 100℃) for 1 h or at 121℃ for 15 min. After treatment, the anti-methicillin-resistant Staphylococcus aureus activity was estimated by the disk diffusion method. All assays were done in triplicate.
- Effect of the ethyl acetate extract on MRSA cell morphology
We also investigated the morphology of MRSA cells exposed to the ethyl acetate extract using transmission electron microscopy. As shown in Fig. 4 , MRSA cell lysis was observed following growth at 37℃ for 24 h with the ethyl acetate extract (320 μg/mL). Several antibiotics, including penicillin and vancomycin, interfere with cell wall synthesis, leading to cell lysis (Barna and Williams, 1984). Based on our results, we propose that Pseudomonas sp. UJ-6 produces a substance that interferes with wall synthesis. However, we strongly believe that the anti-MRSA mechanism of Pseudomonas sp. UJ-6 differs from that of vancomycin since vancomycin was not effective against gram-negative bacteria (Lee et al., 2008a).
- Thermal and pH stability of the ethyl acetate extract
The thermal stability and pH stability of the ethyl acetate extract were also investigated. The extract maintained >95% activity at pH 3.0-8.0, but it exhibited about 80% and 60% activity at pH 9.0 and 10.0, respectively, when the activity at pH 7.0 was defined as 100% ( Fig. 5 A). As shown in Fig. 5 , the extract was highly resistant to thermal stress. The extract retained >95% of its activity after heat treatment for 15 min at 121℃ ( Fig. 5 B). This result suggests that Pseudomonas sp. UJ-6 produces a heat-stable antibiotic, even though most known antibiotics are heat-labile. To further address this issue, the structure of the anti-MRSA compound from the crude extract should be determined. We have isolated several bioactive metabolites from Pseudomonas sp. UJ-6 and reported the anti-MRSA activity of 1-acetyl-beta-carboline, a compound isolated from Pseudomonas sp. UJ-6 (Lee et al., 2013). Currently, we are working to determine the structure of the remaining isolates.
From these results, we anticipate that Pseudomonas sp. UJ-6 can be used to develop a novel, heat-stable, broad-spectrum antibiotic for the treatment of MRSA infections.
This work was supported by Dong-eui University FoundationGrant (2012 Year).
Abad MJ , Bedoya LM , Bermejo P 2011 Marine compounds andtheir antimicrobial activities. In: Science against Microbial Pathogens:Communicating Current Research and Technological Advances.Mendez-Vilas A, ed. Formatex Research Badajoz, ES 1293 - 1306
Acebal C , Alcazar R , Cañedo LM , de la Calle F , Rodriguez P , Romero F , Puentes JLF 1998 Two marine Agrobacterium producers ofsesbanimide antibiotics. J Antibiot 51 64 - 67    DOI : 10.7164/antibiotics.51.64
Acebal C , Cañedo LM , Puentes JLF , Baz JP , Romero F , de la Calle F , Grávalos MDG , Rodriguez P 1999 Agrochelin, a new cytotoxicantibiotic from a marine Agrobacterium: taxonomy, fermentation,isolation, physicochemical properties and biological activity. J Antibiot 52 983 - 987    DOI : 10.7164/antibiotics.52.983
Barna JCJ , Williams DH 1984 The structure and mode of actionof glycopeptide antibiotics of the vancomycin group. Annu Rev Microbiol 38 339 - 357    DOI : 10.1146/annurev.mi.38.100184.002011
Barsby T , Kelly MT , Gagné SM , Andersen RJ 2001 Bogorol A producedin culture by a marine Bacillus sp. reveals a novel templatefor cationic peptide antibiotics. Org Lett 3 437 - 440    DOI : 10.1021/ol006942q
Biabani MAF , Laatsch H , Helmke E , Weyland H 1997 δ-Indomycinone: a new member of pluramycin class of antibioticsisolated from marine Streptomyces sp. J Antibiot 50 874 - 877    DOI : 10.7164/antibiotics.50.874
Eom SH , Park JH , Yu DU , Choi JI , Choi JD , Lee MS , Kim YM 2011 Antimicrobial activity of brown alga Eisenia bicyclis againstmethicillin-resistant Staphylococcus aureus. Fish Aquat Sci 14 251 - 256
Eom SH , Kim DH , Lee SH , Yoon NY , Kim TH , Chung YH , Kim SB , Kim YM , Kim HW , Lee MS , Kim YM 2012 In vitroantibacterial activity and synergistic antibiotic effects of phlorotanninsisolated from Eisenia bicyclis against methicillin-resistantStaphylococcus aureus. Phytother Res. Advanced online publication.    DOI : 10.1002/ptr.4851
Gerard JM , Haden P , Kelly MT , Andersen RJ 1999 Loloatins A-D,cyclic decapeptide antibiotics produced in culture by a tropical marine bacterium. J Nat Prod 62 80 - 85    DOI : 10.1021/np980219f
Hanaki H , Labischinski H , Inaba T , Kondo N , Murakami H , Hiramatsu K. 1998 Increase in glutamine-non-amidated muropeptidesin the peptidoglycan of vancomycin-resistant Staphylococcus aureusstrain Mu50. J Antimicrob Chemother 42 315 - 320    DOI : 10.1093/jac/42.3.315
Hiramatsu K , Hanaki H , Ino T , Yabuta K , Oquri T , Tenover FC 1997 Methicillin-resistant Staphylococcus aureus clinical strainwith reduced vancomycin susceptibility. J Antimicrob Chemother 40 135 - 136    DOI : 10.1093/jac/40.1.135
Hughes CC , Prieto-Davo A , Jensen PR , Fenical W 2008 The marinopyrroles,antibiotics of an unprecedented structure class from amarine Streptomyces sp Org Lett 10 629 - 631    DOI : 10.1021/ol702952n
Imamura N , Nishijima M , Takadera T , Adachi K , Sakai M , Sano H 1997 New anticancer antibiotics, pelagiomicins produced bya new marine bacterium Pelagiobacter variabilis. J Antibiot 50 8 - 12    DOI : 10.7164/antibiotics.50.8
Isnansetyo A , Kamei Y 2003 MC21-A, a bactericidal antibioticproduced by a new marine bacterium, Pseudoalteromonas phenolicasp. nov. O-BC30T, against methicillin-resistant Staphylococcusaureus. Antimicrob Agents Chemother 47 480 - 488    DOI : 10.1128/AAC.47.2.480-488.2003
Isnansetyo A , Kamei Y 2009 Anti-methicillin-resistant Staphylococcusaureus (MRSA) activity of MC21-B, an antibacterialcompound produced by the marine bacterium Pseudoalteromonasphenolica O-BC30T. Int J Antimicrob Agents 34 131 - 135    DOI : 10.1016/j.ijantimicag.2009.02.009
Jevons MP 1961 “Celbenin”-resistant Staphylococci. Br Med J 1 124 - 125    DOI : 10.1136/bmj.1.5219.124-a
Kamei Y , Isnansetyo A 2003 Lysis of methicillin-resistant Staphylococcusaureus by 2,4-diacetylphloroglucinol produced by Pseudomonassp. AMSN isolated from a marine alga. Int J Antimicrob Agents 21 71 - 74    DOI : 10.1016/S0924-8579(02)00251-0
Keller S , Nicholson G , Drahl C , Sorensen E , Fiedler HP , Süssmuth RD 2007 Abyssomicins G and H and atrop-abyssomicin C fromthe marine Verrucosispora strain AB-18-032. J Antibiot 60 391 - 394    DOI : 10.1038/ja.2007.54
Kwon HC , Kauffman CA , Jensen PR , Fenical W 2006 MarinomycinsA-D, antitumor-antibiotics of a new structure class from a marineactinomycete of the recently discovered genus “Marinispora”. J Am Chem Soc 128 1622 - 1632    DOI : 10.1021/ja0558948
Lee DH , Palermo B , Chowdhury M 2008a Successful treatment ofmethicillin-resistant Staphylococcus aureus meningitis with daptomycin. Clin Infect Dis 47 588 - 590    DOI : 10.1086/590257
Lee DS , Kang MS , Hwang HJ , Eom SH , Yang JY , Lee MS , Lee WJ , Jeon YJ , Choi JS , Kim YM 2008b Synergistic effect betweendieckol from Ecklonia stolonifera and β-lactams against methicillin-resistant Staphylococcus aureus. Biotechnol Bioprocess Eng 13 758 - 764    DOI : 10.1007/s12257-008-0162-9
Lee DS , Eom SH , Jeong SY , Shin HJ , Je JY , Lee EW , Chung YH , Kim YM , Kang CK , Lee MS 2013 Anti-methicillin-resistantStaphylococcus aureus (MRSA) substance from the marine bacteriumPseudomonas sp. UJ-6. Environ Toxicol Pharmacol 35 171 - 177    DOI : 10.1016/j.etap.2012.11.011
Levy SB 2005 Antibiotic resistance-the problem intensifies. Adv Drug Deliv Rev 57 1446 - 1450    DOI : 10.1016/j.addr.2005.04.001
Macherla VR , Liu J , Sunga M , White DJ , Grodberg J , Teisan S , Lam KS , Potts BCM 2007 Lipoxazolidinones A, B, and C: antibacterial4-oxazolidinones from a marine actinomycete isolates from aGuam marine sediment. J Nat Prod 70 1454 - 1457    DOI : 10.1021/np0702032
Martín J , da S Sousa T , Crespo G , Palomo S , González I , Tormo JR , de la Cruz M , Anderson M , Hill RT , Vicente F , Genilloud O , Reyes F 2013 Kocurin, the true structure of PM181104, an antimethicillin-resistant Staphylococcus aureus (MRSA) thiazolylpeptide from the marine-derived bacterium Kocuria palustris. Mar Drugs 11 387 - 398    DOI : 10.3390/md11020387
McArthur KA , Mitchell SS , Tsueng G , Rheingold A , White DJ , Grodberg J , Lam KS , Potts BCM 2008 Lynamicins A-E, chlorinatedbisindole pyrrole antibiotics from a novel marine actinomycete. J Nat Prod 71 1732 - 1737    DOI : 10.1021/np800286d
Micek ST 2007 Alternatives to vancomycin for the treatment of methicillin-resistant Staphylococcus aureus infections. Clin Infect Dis 45 S184 - S190    DOI : 10.1086/519471
National Committee for Clinical Laboratory Standards (NCCLS). 2004 Method for Dilution Antimicrobial Susceptibility Testing for Bacteria That Grow Aerobically: Approved Standard. NCCLS Wayne, PA, US. NCCLS Document M7-A6 7th ed.
Pusecker K , Laatsch H , Helmke E , Weyland H 1997 Dihydrophencomycinmethyl ester, a new phenazine derivative from a marineStreptomycete. J Antibiot 50 479 - 483    DOI : 10.7164/antibiotics.50.479
Rahman H , Austin B , Mitchell WJ , Morris PC , Jamieson DJ , Adams DR , Spragg AM , Schweizer M 2010 Novel anti-infectivecompounds from marine bacteria. Mar Drugs 8 498 - 518    DOI : 10.3390/md8030498
Schaberg DR , Culver DH , Gaynes RP 1991 Major trends in the microbialetiology of nosocomial infection. Am J Med 91 72S - 75S    DOI : 10.1016/0002-9343(91)90346-Y
Shiozawa H , Shimada A , Takahashi S 1997 Thiomarinols D, E, Fand G, new hybrid antimicrobial antibiotics produced by a marinebacterium: isolation, structure, and antimicrobial activity. J Antibiot 50 449 - 452    DOI : 10.7164/antibiotics.50.449
Totsuka K , Shiseki M , Kikuchi K , Matsui Y 1999 Combined effectsof vancomycin and imipenem against methicillin-resistantStaphylococcus aureus (MRSA) in vitro and in vivo. J Antimicrob Chemother 44 455 - 460    DOI : 10.1093/jac/44.4.455
Witte W 1999 Antibiotic resistance in gram-positive bacteria: epidemiologicalaspects. J Antimicrob Chemother 44 1 - 9    DOI : 10.1093/jac/44.suppl_1.1
Woodford N 2005 Biological counterstrike: antibiotic resistance mechanismsof Gram-positive cocci. Clin Microbiol Infect 11 2 - 21    DOI : 10.1111/j.1469-0691.2005.01140.x