In vitro activities of the lichen secondary

metabolites vulpinic acid, (+)-usnic acid, and

(-)-usnic acid against aerobic and anaerobic
microorganisms.
M Lauterwein, M Oethinger, K Belsner, T Peters and R Marre
Antimicrob. Agents Chemother. 1995, 39(11):2541. DOI:
10.1128/AAC.39.11.2541.

Downloaded from http://aac.asm.org/ on August 18, 2014 by UNIV OF YORK

Updated information and services can be found at:
http://aac.asm.org/content/39/11/2541

These include:
CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles
cite this article), more»

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml

To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1995, p. 2541–2543 Vol. 39, No. 11
0066-4804/95/$04.0010
Copyright q 1995, American Society for Microbiology

NOTES
In Vitro Activities of the Lichen Secondary Metabolites Vulpinic Acid,
(1)-Usnic Acid, and (2)-Usnic Acid against Aerobic
and Anaerobic Microorganisms
MICHAEL LAUTERWEIN,1 MARGRET OETHINGER,2 KLAUS BELSNER,1
THIES PETERS,1 AND REINHARD MARRE2*

Downloaded from http://aac.asm.org/ on August 18, 2014 by UNIV OF YORK

Department of Pharmacy and Pharmacology of Natural Compounds1 and Department for Medical
Microbiology and Hygiene,2 University of Ulm, Ulm, Germany
Received 31 May 1995/Returned for modification 7 June 1995/Accepted 15 August 1995

Secondary metabolites of different species of lichen were tested for their activities against a variety of
microbial species. While gram-negative rods and fungi were not inhibited by these compounds, Staphylococcus
aureus, Enterococcus faecalis, Enterococcus faecium, and some anaerobic species (Bacteroides and Clostridium
species) were susceptible at the concentrations tested. Vulpinic acid generally was less active than usnic acid,
regardless of its stereochemistry. The susceptibility to usnic acid was not impaired in clinical isolates of S.
aureus resistant to methicillin and/or mupirocin.

The intensive use of antibiotics has selected for antibiotic
resistance factors and facilitated the spread of multiply resis-
tant microorganisms. Some of the resistant strains such as
Corynebacterium jeikeium or Mycobacterium avium belong to
species with low pathogenicities; however, highly clinically rel-
evant species such as Staphylococcus aureus (4, 5, 11), Entero-
coccus faecalis, and Enterococcus faecium (1) also acquired
resistance to additional antibiotics. The resulting strains limit
the spectrum of therapeutically active antibiotics drastically.
Therefore, there is a continuous need for modifications to
established anti-infective compounds and for the detection of
new chemicals. The search for new chemical structures with
antimicrobial activity traditionally requires testing of large bat-
teries of different microbial products. A more selected way of
finding new compounds could be the use of experiences of
traditional medicine. As early as the 18th century B.C. in Egypt
(16), lichen extracts had been used topically. Pharmacologic
research in the beginning of the antibiotic era in the 1950s
defined usnic acid and related products as the active com-
pounds in lichens (2, 6–8, 12, 15). Preliminary reports revealed
an activity mainly against gram-positive cocci. However, most
of these experiments were carried out more than 40 years ago,
so that the data obtained are not comparable to standardized
test results. We therefore characterized the antibacterial activ-
ities of three acids obtained from lichen.
The antimicrobial activities of the lichen substances (1)-
usnic acid (Fig. 1) (Sigma Chemical Co., St. Louis, Mo.) and
(2)-usnic acid (Fig. 1) of Cladonia stellaris (Opiz) Tonzar &
Viezda (Sigma Chemical Co.) and vulpinic acid (Fig. 2) of
Letharia vulpina (L.) Hue (Sigma Chemical Co.) were tested
against selected aerobic and anaerobic microorganisms by
standardized methodology (13, 14). After ruling out the intrin-
sic activity of tetrahydrofuran (Fluka Chemie AG, Buchs, Swit-
* Corresponding author. Mailing address: Department for Medical
Microbiology and Hygiene, University of Ulm, Robert-Koch-Str. 8,
89081 Ulm, Germany. Phone: 0049 731 502 4601. Fax: 0049 731 502
4619.
zerland), tetrahydrofuran (0.2%, by volume) was used as the
solvent mediator for the lichen compounds. Because of the
limited solubilities of the tested compounds in water-based
nutrient broths (cation-adjusted Mueller-Hinton broth for
aerobic bacteria, Sabouraud broth for Candida species, and
Schaedler broth for anaerobic bacteria) (9, 13, 14), the maxi-
mum achievable concentration in broth was 32 mg/ml. Stock
solutions were filter sterilized as described by Ghione et al. (3)
and shock frozen at 2708C. This procedure did not affect the
antimicrobial potencies of the compounds.
MICs were determined by the microdilution method de-
scribed previously (9, 13, 14) by using an inoculum of 5 3 105
CFU/ml. Mupirocin was a gift of SmithKline Beecham, Mu-
nich, Germany. It was considered susceptible at a breakpoint
of 4 mg/ml. Quality control strains were Staphylococcus aureus
ATCC 29213, Enterococcus faecalis ATCC 29212, Saccharomy-
ces cerevisiae ATCC 9763, and Bacteroides fragilis ATCC 25285.
For performance controls, strains for which MICs were known
according to the National Committee for Clinical Laboratory
Standards (13, 14) were used. Determination of the MBC was
carried out as described previously (10). Representative strains
of enterococci and staphylococci for which the MICs of the two
optic antipodal compounds of usnic acid were 4 mg/ml were
chosen. The MBC was defined as the lowest concentration that
reduced the original inoculum by $99.9%.
In order to get a general impression of the in vitro activity,
the lichen compounds were tested against the following strains
from the American Type Culture Collection: Escherichia coli
ATCC 25922, Enterococcus faecalis ATCC 29212, Haemophi-
lus influenzae ATCC 49247, Pseudomonas aeruginosa ATCC
27853, Staphylococcus aureus ATCC 29213, Streptococcus
pneumoniae ATCC 49619, Candida albicans ATCC 2091, Sac-
charomyces cerevisiae ATCC 9763, Clostridium perfringens
ATCC 13124, Bacteroides thetaiotaomicron ATCC 29741, Bac-
teroides vulgatus ATCC 8482, and Bacteroides fragilis ATCC
25285. These experiments showed that lichen compounds did
not inhibit gram-negative rods or fungi at concentrations of
#32 mg/ml (data not shown) but were active against staphylo-

2541
2542 NOTES ANTIMICROB. AGENTS CHEMOTHER.

FIG. 1. Structure of usnic acid (the chiral center is indicated by an aster-

FIG. 2. Structure of vulpinic acid.
isk).

cocci, enterococci, and anaerobic bacteria (Tables 1 and 2), acid was not noted (Table 2). In accordance with a preliminary

Downloaded from http://aac.asm.org/ on August 18, 2014 by UNIV OF YORK

confirming earlier qualitative data (3, 6, 7, 12, 15). Therefore, report (3, 15), usnic acid had a higher level of antimicrobial
in a second series of experiments the following recent clinical activity, with small but consistent differences in its effectiveness
isolates were tested: Bacteroides ruminicola subsp. brevis (n 5 against different species. Again, resistance to methicillin or
1), Bacteroides loescheii (n 5 1), Propionibacterium acnes (n 5 mupirocin did not interfere with susceptibility to usnic acid.
2), methicillin-susceptible Staphylococcus aureus (MSSA; n 5 Except for a slight difference in Enterococcus faecalis, the optic
30), methicillin-resistant, mupirocin-susceptible Staphylococ- antipodes of usnic acid did not differ in their antimicrobial
cus aureus (n 5 20), methicillin-resistant, mupirocin-resistant activities, confirming the data of Stoll et al. (15). However, in
Staphylococcus aureus (n 5 8), Enterococcus faecalis (n 5 29),
and Enterococcus faecium (n 5 13). Three methicillin-resistant
Staphylococcus aureus strains were obtained from the Depart-
TABLE 2. In vitro activities of vulpinic acid, (1)-usnic acid, and
ment of Microbiology of the University of Lübeck (S. Gater- (2)-usnic acid against clinical isolates of Staphylococcus aureus
mann). The microorganisms were stored frozen in the Micro- and Enterococcus
bank system (Mast, Germany) at 2708C until they were tested.
The clinical isolates generally were inhibited by the lichen Species Antimicrobial MIC (mg/ml)b
substances at the concentrations tested, irrespective of their (no. of isolates)a agent Range 50% 90%
other resistance phenotypes (Table 2). Vulpinic acid was less
active than usnic acid against all aerobic cocci and anaerobic Enterococcus faecalis Vulpinic acid 16–.16 .16 .16
rods tested. Enterococcus species tended to be more resistant (29)
(1)-Usnic acid 4–8 4 8
to vulpinic acid than Staphylococcus aureus. An association
(2)-Usnic acid 4–16 8 8
between susceptibility to methicillin or mupirocin and vulpinic Ampicillin 1–8 2 8

Enterococcus faecium Vulpinic acid 16–32 .16 .16

TABLE 1. In vitro activities of vulpinic acid, (1)-usnic acid, and (13)
(2)-usnic acid against anaerobic rods (1)-Usnic acid 4–16 16 16
(2)-Usnic acid 4–16 16 16
Antimicrobial MIC Ampicillin 0.5–16 16 .16
Species (no. of isolates)
agent (mg/ml)
Staphylococcus aureus Vulpinic acid 4–.16 16 .16
Clostridium perfringens ATCC 13124 Vulpinic acid 16 (MSSA, mps) (30)
(1)-Usnic acid 4 (1)-Usnic acid 2–.16 8 16
(2)-Usnic acid 4 (2)-Usnic acid 2–16 8 16
Ampicillin 0.125–16 8 16
Bacteroides thetaiotaomicron ATCC 29741 Vulpinic acid .16 Gentamicin 0.125–64 0.5 4
(1)-Usnic acid 4 Mupirocin 0.03–0.25 0.06 0.25
(2)-Usnic acid 8
Staphylococcus aureus Vulpinic acid 8–.16 16 .16
Bacteroides vulgatus ATCC 8482 Vulpinic acid 16 (MRSA, mps) (20)
(1)-Usnic acid 4 (1)-Usnic acid 4–16 8 16
(2)-Usnic acid 8 (2)-Usnic acid 4–16 8 16
Ampicillin 4–.16 .16 .16
Bacteroides fragilis ATCC 25285 Vulpinic acid 16 Gentamicin 0.5–128 128 128
(1)-Usnic acid 2 Mupirocin 0.03–0.12 0.06 8
(2)-Usnic acid 1
Staphylococcus aureus Vulpinic acid 8–.16 16 .16
Bacteroides ruminicola subsp. brevis (1) Vulpinic acid .16 (MRSA, mpr) (8)
(1)-Usnic acid 8 (1)-Usnic acid 4–16 8 8
(2)-Usnic acid 16 (2)-Usnic acid 4–16 8 8
Ampicillin .16 .16 .16
Bacteroides loeschii (1) Vulpinic acid 16 Gentamicin 32–128 32 128
(1)-Usnic acid 2 Mupirocin 8–16 16 16
(2)-Usnic acid 2
a
Abbreviations: MSSA, methicillin-susceptible Staphylococcus aureus; mps
Propionibacterium acnes (2) Vulpinic acid 4 mupirocin susceptible (MIC, #4 mg/ml); MRSA, methicillin-resistant Staphylo-
(1)-Usnic acid 2 coccus aureus; mpr, mupirocin resistant (MIC, .4 mg/ml).
b
(2)-Usnic acid 2 50% and 90%, MICs at which 50 and 90% of isolates, respectively, are
inhibited.
VOL. 39, 1995
Streptococcus mutans Ghione et al. (3) found that (1)-usnic
acid was significantly more active than (2)-usnic acid.
Usnic acid did not show any bactericidal activity at the con-
centrations tested. In two strains of enterococci, a 98% inhi-
bition of growth was seen at 16 mg/ml for both optic antipodes;
in staphylococci, only 65% inhibition of growth was achieved.
Stoll et al. (15) reported a bacteriostatic effect against staph-
ylococci but a bactericidal effect against Mycobacterium tuber-
culosis in vitro (7, 15), which could not be confirmed in exper-
iments in animals by other investigators (8).
To our knowledge, our study is the first published investiga-
tion on the antimicrobial activities of three secondary metab-
olites from lichens determined by applying standardized meth-
odology. Taken together, the most important finding of our
study was that a compound chemically unrelated to other
known antibiotics shows antibacterial activity against relevant
pathogens and is active against Staphylococcus aureus, regard-
less of the strain’s resistance to methicillin or mupirocin. Be-
cause of the lipophilic properties of the compound, higher
concentrations can be expected in ointments; however, de-
tailed data on solubility have not been published. Thus, a
possible application of the usnic acids or related compounds
might be the topical eradication of nosocomially acquired
Staphylococcus aureus isolates. The lack of bactericidal activity
may limit its use.
We thank Hermann Muhle, Spezielle Botanik, University of Ulm,
for providing information on lichen substances.
REFERENCES
1. Courvalin, P. 1990. Resistance of enterococci to glycopeptides. Antimicrob.
Agents Chemother. 34:2291–2296.
2. Culberson, C. F., W. L. Culberson, and A. Johnson. 1977. Usnic acids and
NOTES 2543
related products, p. 49–51. In Second supplement to chemical and botanical
guide to lichen products. University of North Carolina Press, Chapel Hill.
3. Ghione, M., D. Parrello, and L. Grasso. 1988. Usnic acid revisited, its activity
on oral flora. Chemioterapia 7:302–305.
4. Johnson, A. P., A. H. C. Uttley, N. Woodford, and R. C. George. 1990.
Resistance to vancomycin and teicoplanin: an emerging clinical problem.
Clin. Microbiol. Rev. 3:280–291.
5. Kaatz, G. W., S. M. Seo, N. I. Dorman, and S. A. Lerner. 1990. Emergence
of teicoplanin resistance during therapy of Staphylococcus aureus endocar-
ditis. J. Infect. Dis. 162:103–108.
6. Klosa, J. 1950. Antibiotika aus Flechten und Ihre therapeutische Verwend-
ungsmöglichkeiten. Med. Monatsschr. 11:816–820.
7. Klosa, J. 1951. Über die antibiotische Wirkung von Flechtenstoffen. Z.
Physiol. Chem. 287:195–204.
8. Klosa, J. 1953. Chemische Konstitution und antibiotische Wirkung der
Flechtenstoffe. Pharmazie 8:435–442.
9. Lorian, V. (ed.). 1991. Antifungal drugs—susceptibility testing, p. 218–234.
Downloaded from http://aac.asm.org/ on August 18, 2014 by UNIV OF YORK
In Antibiotics in laboratory medicine, 3rd ed. The Williams & Wilkins Co.,
Baltimore.
10. Lorian, V. (ed.). 1991. Susceptibility testing of antimicrobials in liquid me-
dia—the minimum bactericidal concentration (MBC), p. 96–98. In Antibi-
otics in laboratory medicine, 3rd ed. The Williams & Wilkins Co., Baltimore.
11. Maple, P. A., J. M. T. Hanuktib-Miller and W. Brumfitt. 1989. World-wide
antibiotic resistance in methicillin-resistant Staphylococcus aureus. Lancet
ii:537–540.
12. Möse, J. R. 1955. Zur antibakteriellen Wirksamkeit der Usninsäure. Arz-
neim.-Forsch. 5:510–513.
13. National Committee for Clinical Laboratory Standards. 1990. Methods for
antimicrobial susceptibility testing of anaerobic bacteria. Document M11-
A2, vol. 10, no. 15. National Committee for Clinical Laboratory Standards,
Villanova, Pa.
14. National Committee for Clinical Laboratory Standards. 1993. Methods for
dilution antimicrobial susceptibility tests for bacteria that grow aerobically.
Document M100-S4, vol. 14. National Committee for Clinical Laboratory
Standards, Villanova, Pa.
15. Stoll, A., A. Brack, and J. Renz. 1950. Die Wirkung von Flechtenstoffen auf
Tuberkelbakterien und auf einige andere Mikroorganismen. Schweiz. Z.
Allgem. Pathol. Bakteriol. 13:729–751.
16. Vartia, K. O. 1973. Antibiotics in lichens, p. 547–561. In V. Ahmadjian and
M. E. Hale (ed.) The lichens. Academic Press, Inc., New York.