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DOI: 10.1055/s-2004-832653
Potent Antibacterial Activity of Halogenated Compounds against Antibiotic-Resistant Bacteria
Dr. Charles S. Vairappan
Laboratory of Marine Natural Product Chemistry
Borneo Marine Research Institute
Universiti Malaysia Sabah
88999 Kota Kinabalu
Sabah
Malaysia
Phone: +60-88-320000 (ext. 2587)
Fax: +60-88-320261
Email: csv@ums.edu.my
Publication History
Received: February 6, 2004
Accepted: May 15, 2004
Publication Date:
18 November 2004 (online)
Abstract
Common Gram-positive clinical pathogens are showing an increasing trend for resistance to conventional antimicrobial agents. New drugs with potent antibacterial activities are urgently needed to remediate this problem. Halogenated compounds isolated from several species of the red algae genus Laurencia were examined for their antibacterial activity against 22 strains of human pathogenic bacteria, 7 strains of which were antibiotic-resistant bacteria. Four phenolic sesquiterpenes and a polybrominated indole showed wide spectra of antibacterial activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae, and vancomycin-resistant Enterococcus faecalis and E. faecium (VRE). In addition, laurinterol and allolaurinterol displayed potent bactericidal activity against three strains of MRSA at 3.13 μg mL-1, and three strains of vancomycin-susceptible Enterococcus, at 3.13 μg mL-1 and 6.25 μg mL-1, respectively.
The emergence of antibiotic-resistant bacteria is often regarded as an inevitable phenomenon and its increasing incidence has brought a new sense of urgency to the discovery and development of new and potent antibacterial drugs. Common clinical Gram-positive bacterial pathogens have shown remarkable ability to develop resistance to antimicrobial agents. Recent discoveries have shown that resistance has reached a point where some infections evade all available antibiotics. Resistance to treatment for methicillin-resistant Staphylococcus aureus (MRSA) was regarded as a serious problem but now even vancomycin (VCM) has been found to be inactive against an increasing number of clinical bacteria [1], [2]. Regardless of the amount of investment involved in antibiotic discovery, there seems to be a finite life for every antibiotic in terms of being able to use it without the fear of resistance. Resistant Gram-positive bacteria can be regarded as the prominent cause of serious hospital infections [3]. Hence, the search for new antibiotics is inevitable and must go on to ensure an effective arsenal to battle human pathogenic bacteria that have become resistance to our present array of drugs. In an attempt to search for potential antibacterial drugs, we looked at halogenated metabolites of red algae genus Laurencia. Halogenated secondary metabolites isolated from Laurencia species are known for their structural diversity with unique biological activities [4]. These compounds have been known as potent antibacterial agents against terrestrial [5], [6], [7], [8], [9] and marine [10], [11] bacteria. Some of them have been proven to be potent against human pathogenic bacteria derived from clinical patients [12].
The present study was carried out to establish the potentials of marine halogenated metabolites as possible ”lead pharmaceutical compounds” in an effort to facilitate discovery of new drugs and combat the increase in antibacterial resistant bacteria. Hence, we first screened the antibacterial activity of eight halogenated compounds isolated from five species of Laurencia against 22 strains of human pathogenic bacteria (seven strains of antibiotic-resistant bacteria). Subsequently, the potency of highly active compounds was evaluated based on the respective compound’s bactericidal activity.
A total of 8 halogenated secondary metabolites were isolated from 5 Laurencia species collected from Japanese and Malaysian waters. Laurinterol (1) and isolaurinterol (2) are major characteristic metabolites of L. okamurae [13], one of the well studied Japanese Laurencia species. Allolaurinterol (10-bromo-7-hydroxylaurene) (3) was previously reported as a constituent of Laurencia filiformis [14] and Laurencia subopposita [6]. 10-Bromo-7-hydroxylaurene isolated in this study contained a small amount of inseparable 10,11-dibromo-7-hydroxylaurene (3′). While, cupalaurenol (4) reported here was initially isolated from the sea hare Aplysia dactylomela collected from Kohama Island, Okinawa Prefecture [15]. In total 4 compounds were isolated from Malaysian Laurencia, elatol (5) and iso-obtusol (6) were first isolated from L. elata [16] and L. obtusa [17], respectively. Polybrominated indoles, 2,3,5,6-tetrabromoindole (7) and 1-methyl-2,3,5,6-tetrabromoindole (8) were previously isolated from L. brongniartii (7). In an effort to provide a broad indication of the biological activity of the isolated metabolites, antibacterial effects were evaluated against 22 strains of human pathogenic bacteria including 7 strains of antibiotic-resistant bacteria. The results of the antibacterial bioassay are reported in Table [1] (for structures, see Fig. [1]).
Out of the 8 halogenated compounds tested, compounds 1, 2, 3, 4 and 7 showed wide spectra of activity against Gram-positive bacteria including MRSA, penicillin-resistant Streptococcus pneumoniae and VCM-resistant Enterococcus faecalis and E. faecium. Their activity against VCM-susceptible strains was overwhelming, less than that of VCM. These compounds also showed antibacterial activity against all enterococcal strains tested, regardless of VCM-resistant pattern. However, these compounds did not exhibit antibacterial activity against Gram-negative pathogens except against Moraxella catarrhalis. But, two halochamigrene derivatives, 5 and 6, and an N-methylindole (8) were inactive against the tested pathogens. On the other hand, two phenolic sesquiterpenes, laurinterol (1) and allolaurinterol (10-bromo-7-hydroxylaurene) (3) demonstrated potent bactericidal activity against methicillin-resistant S. aureus (MRSA) at a concentration of 2 × MIC value (6.25 μg/mL), and their activity was more potent than that of VCM. Both compounds 1 and 3 were also bactericidal against VCM-susceptible E. faecium, although VCM was bacteriostatic. Prior to this study, halogenated compounds used in this investigation were tested against environmental bacteria and were found to have potent antibacterial activities [10] (and unpublished data). Compounds 5 and 6 even showed potent antibacterial activities at the concentration of 30 μg disc-1 against freshly isolated human pathogenic bacteria (non-resistance strains) but were inactive against the bacteria tested in this study [12]. Thus, a comparison of antibacterial activity for compounds 5 and 6 conducted in these studies could clearly illustrate seriousness of bacterial resistance and the urgent need to find alternative drugs.
However, the presence of strong bactericidal activity in compounds 1 and 3 promises some hope in our desperate attempt to discover potent drugs. Hence, halogenated metabolites derived from marine red algal genus Laurencia may have some potential to be applied as effective drugs for conquering multi-antibiotic resistant bacteria, such as methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA) and vancomycin-resistant E. faecium (VRE).
Fig. 1 Structures of halogenated compounds 1 - 8 from Laurencia species.
Materials and Methods
Halogenated metabolites were obtained from the red algal genus Laurencia of the Japanese or Malaysian waters. Structures of the tested compounds are illustrated in Fig. [1]. Compounds 1 (laurinterol) and 2 (isolaurinterol) were isolated from L. okamurae Yamada collected at Oshoro Bay, Hokkaido, Japan [13]. Compounds 3 (allolaurinterol; 10-bromo-7-hydroxylaurene) and 4 (cupalaurenol) were isolated from L. majuscula (Harvey) Lucas and L. venusta Yamada, respectively, collected at Tanegashima Island, Kagoshima Prefecture, Japan [18]. Two halochamigrene derivatives, compounds 5 (elatol) and 6 (iso-obtusol) were isolated from L. majuscula (Harvey) Lucas collected at several locations in Malaysia, e. g., Pulau Bankawan, Sandakan, Sabah, Malaysia [10]. Moreover, two polybrominated indoles, compounds 7 (2,3,5,6-tetrabromoindole) and 8 (1-methyl-2,3,5,6-tetrabromoindole) were isolated from L. similes Nam et Saito collected at Pulau Gaya, Sabah, Malaysia [19]. Isolation protocols and structural elucidation of the compounds used in this study are elaborated in the respective references as stated above. Purities of the compounds were confirmed by 1H-NMR; compounds 1, 2, 4, 5, 6, 7 and 8 were more than 99 % pure, while compound 3 (95 % pure) was present as an inseparable mixture with compound 3′ (5 %).
Antibacterial susceptibility tests and minimum inhibitory concentration (MIC) determinations were conducted for the Laurencia-derived compounds and vancomycin (VCM) by the agar dilution method against Gram-positive (8 species 15 strains) and Gram-negative (7 species 7 strains) bacteria. Details of the bacteria are given in Table [1]. Bactericidal activity was studied by means of a time-kill assay using concentrations of 1/2 × , 1 × and 2 × MIC of laurinterol (1) or allolaurinterol (10-bromo-7-hydroxylaurene) (3) with approximate inoculums of 105 CFU/mL of mythically-resistant Staphylococcus aureus SR3637 (MRSA) and VCM-susceptible Enterococcus faecium SR15941.
| Tested Pathogenic Bacteria | Minimum Inhibitory Concentration (MIC; μg/mL) | ||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | VCMe | |
| Staphylococcus aureus 209P JC-1 | 3.13 | 6.25 | 3.13 | 3.13 | 25 | > 50 | 6.25 | > 50 | 0.39 |
| Staphylococcus aureus Smith | 3.13 | 6.25 | 3.13 | 6.25 | 50 | > 50 | 6.25 | > 50 | 0.78 |
| Staphylococcus aureus SR313 (L-MRSA)a | 3.13 | 6.25 | 3.13 | 6.25 | 25 | > 50 | 6.25 | > 50 | 1.56 |
| Staphylococcus aureus SR3626(H-MRSA)a | 3.13 | 6.25 | 3.13 | 6.25 | 50 | > 50 | 3.13 | > 50 | 0.78 |
| Staphylococcus aureus SR3637(H-MRSA)a | 3.13 | 6.25 | 3.13 | 6.25 | 50 | > 50 | 6.25 | > 50 | 1.56 |
| Staphylococcus epidemidis ATCC14990 | 3.13 | 6.25 | 6.25 | 6.25 | > 50 | > 50 | 6.25 | > 50 | 1.56 |
| Staphylococcus haemolyticus ATCC29970 | 3.13 | 6.25 | 6.25 | 6.25 | > 50 | > 50 | 6.25 | > 50 | 1.56 |
| Streptococcus pyogenes C-203 | 1.56 | 3.13 | 1.56 | 3.13 | 25 | > 50 | 3.13 | > 50 | 0.39 |
| Streptococcus pneumoniae Type 1 | 1.56 | 3.13 | 1.56 | 3.13 | 25 | > 50 | 3.13 | > 50 | 0.39 |
| Streptococcus pneumoniae SR16675-PRSPb | 3.13 | 6.25 | 3.13 | 3.13 | 25 | > 50 | 3.13 | > 50 | 0.39 |
| Streptococcus mitis SR16376 | 3.13 | 6.25 | 3.13 | 6.25 | 50 | > 50 | 6.25 | > 50 | 0.78 |
| Enterococcus faecalis SR1004 | 6.25 | 6.25 | 6.25 | 6.25 | > 50 | > 50 | 6.25 | > 50 | 1.56 |
| Enterococcus faecalis ATCC51299(Van B)c | 3.13 | 6.25 | 6.25 | 6.25 | > 50 | > 50 | 6.25 | > 50 | 12.5 |
| Enterococcus faecalis SR7914(Van A)d | 3.13 | 6.25 | 6.25 | 6.25 | > 50 | > 50 | 6.25 | > 50 | > 50 |
| Enterococcus faecium SR7917(Van A)d | 3.13 | 6.25 | 6.25 | 6.25 | 50 | > 50 | 6.25 | > 50 | > 50 |
| Escherichia coli NIHJ JC-2 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 |
| Klebsiella pneumoniae SR1 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 |
| Morganella morganii SR9 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 |
| Enterobacter cloacae ATCC13047 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 |
| Serratia marcescens ATCC13880 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 |
| Pseudomonas aeruginosa ATCC25619 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 | > 50 |
| Moraxella catarrhalis SR11341 | 1.56 | 3.13 | 1.56 | 3.13 | 12.5 | 50 | 1.56 | > 50 | 50 |
| a Methicillin-resistant S. aureus. | |||||||||
| b Penicillin-resistant S. pneumoniae. | |||||||||
| c,d Vancomycin-resistant strain. | |||||||||
| e VCM-Vancomycin. | |||||||||
References
- 1 Panlilio A L, Culver D H, Gaynes R P. et al . Methicillin-resistant S. aureus in US hospitals, 1975 - 1991. Infection Control Hospital Epidemiol. 1992; 13 582-6
- 2 Moellering R C. Vancomycin-resistant enterococci. Clin Infect Dis. 1998; 26 1196-9
- 3 Diekema D J, Jones R N. Oxazolidinone antibiotics. Lancet. 2001; 358 1975-82
- 4 Erickson K L. Marine Natural Products: Chemical and Biological Perspectives. Constituents of Laurencia. In: Scheuer PJ, editor Vol. 5 Academic Press New York; 1983: pp 131-257
- 5 Waraszkiewicz S M, Erickson K L. Halogenated sesquiterpenoids from the Hawaiian marine alga Laurencia nidifica: nidificene and nidifidiene. Tetrahedron Lett 1974: 2003-6
- 6 Wratten S J, Faulkner D J. Metabolites of the red alga Laurencia subopposita . J Org Chem. 1977; 42 3343-9
- 7 Carter G T, Rinehart K L, Li L H, Kuentzel S L, Connor J L. Brominated indoles from Laurencia brongniartii . Tetrahedron Lett 1978: 4479-82
- 8 Caccamese S, Azzolina R, Duesler E N, Paul I C, Rinehart K L. Laurencienyne, a new acetylenic cyclic ether from the marine red alga Laurencia obtusa . Tetrahedron Lett. 1980; 21 2299-302
- 9 König G M, Wright A W. Sesquiterpene content of the antibacterial dichloromethane extract of the marine red alga Laurencia obtusa . Planta Medica. 1997; 63 186-7
- 10 Vairappan C S, Daitoh M, Suzuki M, Abe T, Masuda M. Antibacterial halogenated metabolites from the Malaysian Laurencia species. Phytochemistry. 2001; 58 291-7
- 11 Vairappan C S, Suzuki M, Abe T, Masuda M. Halogenated metabolites with antibacterial activity from the Okinawan Laurencia species. Phytochemistry. 2001; 58 517-23
- 12 Vairappan C S. Potent antibacterial activity of halogenated metabolites from Malaysian red algae, Laurencia majuscula (Rhodomelaceae, Ceramiales). Biomol Engineering. 2003; 20 255-9
- 13 Suzuki M, Kurosawa E. Halogenated and non-halogenated aromatic sesquiterpenes from the red algae Laurencia okamurai Yamada. Bull Chem Soc Japan. 1979; 52 3352-4
- 14 Kazlauskas R, Murphyi P T, Quinn R J, Wells R J. New laurene derivatives from Laurencia filiformis . Aust J Chem. 1976; 29 2533-9
- 15 Ichiba T, Higa T. New cuparene-derived sesquiterpenes with unprecedented oxygenation patterns from the sea hare Aplysia dactylomela . J Org Chem. 1986; 51 3364-6
- 16 Sims J J, Lin G HY, Wing R M. Marine natural product X. Elatol, a halogenated sesquiterpene alcohol from the red alga Laurencia elata . Tetrahedron Lett 1974: 3487-90
- 17 Gonzalez A G, Martin J D, Martin V S, Martinez-Ripoli M, Fayos J. X-ray studies of sesquiterpene constituents of the alga L. obtusa leads to structure revision. Tetrahedron Lett 1979: 2717-8
- 18 Masuda M, Kawaguchi S, Abe T, Kawamoto T, Suzuki M. Additional analysis of chemical diversity of the red algal genus Laurencia (Rhodomelaceae) from Japan. Phycol Res. 2002; 50 135-44
- 19 Masuda M, Kawaguchi S, Takahashi Y, Okamoto K, Suzuki M. Halogenated secondary metabolites of Laurencia similis (Rhodomelaceae, Rhodophyta). Botanica Marina. 1999; 42 199-202
Dr. Charles S. Vairappan
Laboratory of Marine Natural Product Chemistry
Borneo Marine Research Institute
Universiti Malaysia Sabah
88999 Kota Kinabalu
Sabah
Malaysia
Phone: +60-88-320000 (ext. 2587)
Fax: +60-88-320261
Email: csv@ums.edu.my
References
- 1 Panlilio A L, Culver D H, Gaynes R P. et al . Methicillin-resistant S. aureus in US hospitals, 1975 - 1991. Infection Control Hospital Epidemiol. 1992; 13 582-6
- 2 Moellering R C. Vancomycin-resistant enterococci. Clin Infect Dis. 1998; 26 1196-9
- 3 Diekema D J, Jones R N. Oxazolidinone antibiotics. Lancet. 2001; 358 1975-82
- 4 Erickson K L. Marine Natural Products: Chemical and Biological Perspectives. Constituents of Laurencia. In: Scheuer PJ, editor Vol. 5 Academic Press New York; 1983: pp 131-257
- 5 Waraszkiewicz S M, Erickson K L. Halogenated sesquiterpenoids from the Hawaiian marine alga Laurencia nidifica: nidificene and nidifidiene. Tetrahedron Lett 1974: 2003-6
- 6 Wratten S J, Faulkner D J. Metabolites of the red alga Laurencia subopposita . J Org Chem. 1977; 42 3343-9
- 7 Carter G T, Rinehart K L, Li L H, Kuentzel S L, Connor J L. Brominated indoles from Laurencia brongniartii . Tetrahedron Lett 1978: 4479-82
- 8 Caccamese S, Azzolina R, Duesler E N, Paul I C, Rinehart K L. Laurencienyne, a new acetylenic cyclic ether from the marine red alga Laurencia obtusa . Tetrahedron Lett. 1980; 21 2299-302
- 9 König G M, Wright A W. Sesquiterpene content of the antibacterial dichloromethane extract of the marine red alga Laurencia obtusa . Planta Medica. 1997; 63 186-7
- 10 Vairappan C S, Daitoh M, Suzuki M, Abe T, Masuda M. Antibacterial halogenated metabolites from the Malaysian Laurencia species. Phytochemistry. 2001; 58 291-7
- 11 Vairappan C S, Suzuki M, Abe T, Masuda M. Halogenated metabolites with antibacterial activity from the Okinawan Laurencia species. Phytochemistry. 2001; 58 517-23
- 12 Vairappan C S. Potent antibacterial activity of halogenated metabolites from Malaysian red algae, Laurencia majuscula (Rhodomelaceae, Ceramiales). Biomol Engineering. 2003; 20 255-9
- 13 Suzuki M, Kurosawa E. Halogenated and non-halogenated aromatic sesquiterpenes from the red algae Laurencia okamurai Yamada. Bull Chem Soc Japan. 1979; 52 3352-4
- 14 Kazlauskas R, Murphyi P T, Quinn R J, Wells R J. New laurene derivatives from Laurencia filiformis . Aust J Chem. 1976; 29 2533-9
- 15 Ichiba T, Higa T. New cuparene-derived sesquiterpenes with unprecedented oxygenation patterns from the sea hare Aplysia dactylomela . J Org Chem. 1986; 51 3364-6
- 16 Sims J J, Lin G HY, Wing R M. Marine natural product X. Elatol, a halogenated sesquiterpene alcohol from the red alga Laurencia elata . Tetrahedron Lett 1974: 3487-90
- 17 Gonzalez A G, Martin J D, Martin V S, Martinez-Ripoli M, Fayos J. X-ray studies of sesquiterpene constituents of the alga L. obtusa leads to structure revision. Tetrahedron Lett 1979: 2717-8
- 18 Masuda M, Kawaguchi S, Abe T, Kawamoto T, Suzuki M. Additional analysis of chemical diversity of the red algal genus Laurencia (Rhodomelaceae) from Japan. Phycol Res. 2002; 50 135-44
- 19 Masuda M, Kawaguchi S, Takahashi Y, Okamoto K, Suzuki M. Halogenated secondary metabolites of Laurencia similis (Rhodomelaceae, Rhodophyta). Botanica Marina. 1999; 42 199-202
Dr. Charles S. Vairappan
Laboratory of Marine Natural Product Chemistry
Borneo Marine Research Institute
Universiti Malaysia Sabah
88999 Kota Kinabalu
Sabah
Malaysia
Phone: +60-88-320000 (ext. 2587)
Fax: +60-88-320261
Email: csv@ums.edu.my
Fig. 1 Structures of halogenated compounds 1 - 8 from Laurencia species.