International Journal of Antimicrobial Agents 18 (2001) 583585

Short communication
The effect of Nepeta cataria extract on adherence and enzyme
production of Staphylococcus aureus

Antonia Nostro *, Maria Angela Cannatelli, Giuseppe Crisa, Vittorio Alonzo

Dipartimento Farmaco-Biologico, Facolta` di Farmacia-Sezione di Microbiologia, Uni6ersita` di Messina, Villaggio Annunziata,
Messina 98168, Italy
Received 23 May 2001; accepted 12 July 2001
Abstract
Nepeta cataria L., commonly known as catnip, is a perennial herb with a considerable folkloric reputation. A diethyl ether
extract of this plant has been shown to have antimicrobial activity against fungi and Gram-positive bacteria. The aim of this work
was to study the activity of N. cataria extract on 44 Staphylococcus aureus strains, some resistant to methicillin, and S. aureus
6538P (American Type Culture Collection) by evaluating the effect of subminimum inhibitory concentrations on coagulase,
DNAse, thermonuclease and lipase production, and on in-vitro adherence. DNAse, thermonuclease and lipase were inhibited by
concentrations equal to 1/2 and 1/4 MIC. A reduction of adherence was also observed. 2001 Elsevier Science B.V. and
International Society of Chemotherapy. All rights reserved.
Keywords: Nepeta cataria; Staphylococcus aureus; Inhibition; Factors virulence
www.ischemo.org
1. Introduction
Nepeta cataria is found from Central Europe to the
Iranian plateaux and in Central Asia, and is common in
North America. The antispasmodic, carminative, seda-
tive, stimulant and tonic properties of this species seem
to account for its use in folk medicine. It is also an
insect repellent and dried leaves and owering tops are
used to prepare a sedative tea [1]. The major con-
stituents of N. cataria appear to be the nepetalactones,
cyclopentanoid monoterpenes with antimicrobial activi-
ties [2]. Other Nepeta species have been studied.
Metabolites of N. leucophylla and N. clarkei have been
screened for antifungal activities and N. hindostana was
found to be more effective than the prevalent synthetic
fungicides used against damping-off diseases [3,4].
This present work evaluated the effect of submini-
mum inhibitory concentrations (sub-MICs) of a diethyl
ether extract of N. cataria on coagulase, DNAse, ther-
monuclease and lipase production by Staphylococcus
aureus and on the in-vitro adherence of the strains.
2. Materials and methods
2.1. Organisms test and plant extract preparation
S. aureus isolates (n=44) were obtained from vari-
ous clinical sources (Table 1). Identication used colony
morphology, Gram staining, coagulase production and
API Staph (API, Biomerieux). Stock cultures were
maintained on nutrient agar slopes at 4 C and subcul-
tured every 2 weeks. S. aureus 6538P ATCC was used
as a control strain.
Powdered N. cataria was extracted with diethyl ether
as described previously [5,6] and the residue was solubi-
lized in dimethylsulphoxide (DMSO; BDH).

This work was presented in part (abstract A-132) at the 100th

General Meeting of the American Society for Microbiology, held in
Los Angeles in May 2000.
* Corresponding author. Tel.: +39-090-3533120; fax: +39-090-
6766438.
E-mail address: atnostro@pharma.unime.it (A. Nostro).
0924-8579/01/$ - $20 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.
PII: S0924- 8579( 01) 00452- 6
A. Nostro et al. / International Journal of Antimicrobial Agents 18 (2001) 583585 584
Table 1
Clinical sources, methicillin-resistance and slime production of S. aureus strains
Origin Number of isolates MSSA MRSA Slime-producer
Strong Medium Weak
10 6 16 2 Nosocomial environment 4
28 Respiratory tract infections 22 6 4 4
32 44 12 14
2.2. Effects on enzymatic acti6ity
The effect of N. cataria extract on some of the
enzymes of S. aureus was estimated as previously re-
ported [6] using extracts incorporated in appropriate
melted agar medium in order to obtain subMIC levels
(from 1000 to 250 mg/l). Ten microlitre of an overnight
broth culture diluted to 10
4
cells was spot-inoculated
onto plates that were then incubated at 37 C for 24 h.
All determinations were performed in duplicate using
DMSO (1% v/v) as control. Inhibition of enzymatic
activity was evaluated as total when the halo was
absent and partial when the halo was 550% compared
with the positive control.
In order to obtain cells free of extract, the cells
grown in the presence of N. cataria subMIC doses were
washed in peptone water (Oxoid) and the pellet was
resuspended in Tryptone Soya broth (TSB; Oxoid).
This suspension was spot-inoculated on appropriate
medium to estimate the enzymatic activities.
2.3. In-6itro adherence
Adherence to smooth surfaces was performed in 96-
well tissue culture plates (Nunclon; Nunc) using the
method of Shadow et al. [7]. The assay used a range of
S. aureus strains (n=14) producing different amounts
of slime. There were three levels of production dened
in an arbitrary manner using absorbance values: strong
(absorbance values from 0.25 to 0.35), medium (0.15
0.25) and weak (0.010.15). SubMIC levels of the
extract were added to overnight cultures grown in TSB
and diluted to 10
5
cfu/ml. Two hundred microlitre of
this solution were dispensed in each plate well and
adherence was measured after 24 h incubation at
37 C. Those organisms that failed to adhere were
removed by aspiration of the supernatant and the wells
were washed four times with PBS (pH 7.2). Organisms
adhering as a lm to the bottom of the wells were xed
with 200 ml Bouins xative (picric acidformalin
(40%) acetic acid; 73:25:2) for 12 h and, after four
washings with PBS, were stained with 0.01% crystal
violet for 510 min. The wells content was suspended
with 10% N-lauroyl-sarkosyne (Sigma) and the A
492
read with BioRad EIA Reader (model 2550). Each
experiment was done in quadruplicate and repeated at
least three times; values were then averaged. All deter-
minations included a control of DMSO 1% (v/v) and
two blanks of TSB and TSB with extract but without
bacteria.
3. Results and discussion
Sub-MIC levels of Helichrysum italicum extract af-
fected some enzymes that contribute to the pathogenic
activities of S. aureus [6]. In this study we found that N.
cataria extract inhibited the enzymatic activities of S.
aureus MRSA and MSSA strains equally. There was
total inhibition of DNAse, thermonuclease and lipase
in the ATCC strain and in most of the test strains but
no reduction of coagulase activity by either dilution
(Table 2). Such inhibition could have been due to direct
or indirect interaction of plant extract and enzymes.
Enzyme activity reappeared when the cells were washed
free of the extract.
Some strains of S. aureus are now resistant to many
antibiotics. Besides extracellular proteins, certain cell
surface adhesins and exocellular materials (slime) pro-
duced by S. aureus may inuence pathogenicity. Iso-
lates of S. aureus causing nosocomial surgical infections
and atopic dermatitis show a disproportionate number
of slime producing strains [8,9]. Slime production has
been associated with strains from surgical infections,
Table 2
Inhibition of enzymatic activity of S. aureus strains (n=44) by N.
cataria extract
N. cataria extract (% of Staphylococcal Enzymes
strains inhibited)
1/4 MIC 1/2 MIC
T
a
P P
b
T
Coagulase
59 24 DNAse 16 73
80 100 Thermonuclease 10
100 15 41 Lipase
a
Total inhibition of activity.
b
Partial inhibition of activity (550% as compared with control).
A. Nostro et al. / International Journal of Antimicrobial Agents 18 (2001) 583585 585
Table 3
Effects of N. cataria extract on S. aureus adherence (n=14). The results represent the means9S.E. of quadruplicate measurements
Range Slime-producer Absorbance
492
(mean values)
Control N. cataria extract
TSB TSB+DMSO1% 1/2 MIC 1/4 MIC
0.32090.015 0.33090.010 Strong 0.04790.002 0.2500.350 0.10590.005
0.19590.025 0.18590.013 0.1500.249 0.03990.004 Medium 0.08790.003
0.10790.010 0.11090.005 0.05090.006 0.08990.004 Weak 0.0100.149
atopic dermatitis and has also been correlated with
resistance to antimicrobial agents [10]. The results of
the adherence experiments suggested that sub-MIC
doses of the extract reduced attachment of S. aureus
strains to polystyrene. Greater inhibition was obtained
with levels of 1/2 MIC than 1/4 MIC and this effect did
not correlate with the initial degree of slime-production
(Table 3).
Previous studies using the diethyl ether extract from
N. cataria showed alkaloids, avonoids and terpenes to
be present [5]. Further studies should be carried out to
isolate and purify the antimicrobial compounds in or-
der to lower the level of an effective dose.
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