Inhibitory Effect of Some Medicinal Plants from Iran on Swarming Motility of
Ali Ghanberi Asad
In order to develop a media to abolish the swarming of proteus without
effecting the growth of other bacteria, the anti-swarming activity of 25 plant
essential oils or extracts were evaluated on standard and clinical isolates of
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli,
Proteus mirabilis, P. vulgaris and P. Penneri,
using standard agar dilution method. The most effective anti-swarming agents
tested were; the mixture containing equal amounts of essential oil of Ferula
gumosa Boiss plus Lavandula officinalis and essential oil of
Zataria multiflora Boiss. Essential oil of Zataria multiflora Boiss
had antibacterial activity and inhibited the growth of many isolates at
concentration of 2%. Whereas the essential oil of Lavandula officinalis
plus Ferula gumosa Boiss although had no antibacterial activity at this
concentration, completely inhibited the swarming behavior of all the proteus
strains tested. This mixture was quite active in the presence of 7.5% blood
as in blood agar plates. Essential oil of Zataria multiflora Boiss
and Lavandula officinalis plus Ferula gumosa Boiss with good
anti-swarming activities and long shelf life, are commercially available and can
be used as an effective anti-swarming agent for preparation of media in clinical
laboratories for the isolation of more fastidious bacteria in a mixed culture
The genus Proteus of the Enterobacteriaceae family consists of four species, P. mirabilis, P. vulgaris, P. penneri, P. hauser and three unnamed genomospecies. These bacteria are widely distributed in natural environments, such as polluted water, soil and manure, where they play an important role in decomposing organic matter of animal origin. Besides the saprophytic mode of life in the natural environments and in the intestine of humans, wild and domestic animals, they are well known opportunistic pathogens and the first three species are involved in urinary tract infections. The most characteristic feature of this genus is the swarming motility[4,5]. This phenomenon or zonal growth is described as the formation of concentric zones of bacterial growth, able to cover the whole surface of solid culture medium. Swarming frequently interferes with the enumeration of other microorganisms in clinical microbiology investigation and enhances the difficulty of isolating organisms from mixed cultures. For example in veterinary microbiology, samples taken from ears of cats and dogs are often contaminated with P. mirabilis and the swarming motility makes it almost difficult or even impossible to isolate single colonies of other bacterial species present in the sample. Many methods and addition of many chemical substances to the agar medium are recommended in order to inhibit the swarming. For example, the use of very dry medium, increasing the concentration of agar, addition of urea, p-nitrophenylglycerol, ethanol, activated charcoal, sodium azide, certain amino acids and ferrous ions[3,5-8]. However most of the works were performed only on reference bacterial strains and the effect of these substances on other pathogens remain largely unknown. Here we describe the inhibitive effect of 25 essential oils or plant extracts on swarming behavior of five strains of P. mirabilis, two strains of P. vulgaris, two strains of P. penneri and the growth of E. coli, P. aeruginosa and S. aureus as the most frequently isolated bacteria in the diagnostic laboratories.
MATERIALS AND METHODS
Bacterial strains: A standard strain of P. mirabilis (ATCC 43071)
was obtained from Mast Diagnostics (France), other strains including Escherichia
coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Pseudomonas
aeruginosa (ATCC 27853) were obtained from Persian Type Culture Collections,
Tehran, Iran. Two strains of P. penneri, two strains of P. vulgaris
and four strains of P. mirabilis were isolated from urinary tract
infections in Kerman, Iran and were identified by standard biochemical methods.
Plant extract and essential oils: Methanolic extracts of Zataria multiflora, Eucalyptus globolus (Labill), Echium ammonium Fisch and Mey and Glycyrrhizia glabra L., were prepared as mentioned earlier other plant extracts and essential oils were obtained in a liquid form from Barij Essence Kashan, Iran (Table 1).
Assay for swarming inhibition: Standard NCCLS agar dilution method was used to test the anti-swarming activity of the plant extracts or essential oils. Stock solutions of dried extracts were made in DMSO and the extracts (0.1 to 0.4 mg mL-1 final concentrations) were added to the Muller Hinton agar (Merck, Germany) after sterilization, at 60°C. Other plant extracts or essential oils (commercial types) were added by the same method to the medium at final concentrations of 0.5 to 2% v/v. Plates were inoculated with 10 μL (105 CFU mL-1) of each organisms, incubated at 37°C and the growth and swarming was monitored 24 h later. Muller Hinton agar without the plant extracts was used as the control of growth and DMSO at its highest concentrations used was tested to see if it had any effect on the growth or the swarming.
From 25 essential oil or methanolic plant extracts used, 13 essential oils
or extracts had no affect either on the swarming or growth of bacterial strains
tested (Table 1). Other essential oils or extracts either
effected the growth or the swarming behavior of some bacterial strains or changed
the pattern of swarming. Myrtus communis L., essential oil (1.5% v/v)
or extract (2 mg mL-1) inhibited the growth of S. aureus,
3 strains of P. mirabilis including the standard strain and one strain
of P. vulgaris but had no effect on the swarming or growth of other tested
bacteria. Essential oil of Gumium cyminum L. (2% v/v), slightly inhibited
the growth of S. aureus, but did not change the swarming (Fig.
1A). In the presence of essential oil of Rosa damascene L., (2% v/v)
P. vulgaris isolates did not swarm (Fig. 1B). Essential
oil of Anethum graveolens L., inhibited the swarming of P. vulgaris
and P. penneri strains plus two strains of P. mirabilis,
other strains swarmed on the medium, forming a turbid membrane on the agar surface
which masked the original spot of bacterial inoculation.
||Evaluation of the effect of native medicinal plants of Iran
used as essential oils (1.25-1.5% W/W) or extracts (0.1- 0.4 mg mL-1)
on swarming or growth of 9 strains of proteus plus standard strains of E.
coli, S. aureus and P. aeruginosa
|Yes: The swarming pattern was changed; Yes+: The
pattern was changed and swarming of some strains was inhibited; No: No difference
with the control
Artemisia sieberi at concentration of 2% (v/v) also inhibited the swarming
of both P. vulgaris strains, one strain of P. penneri and two
strains of P. mirabilis. Extract of Eucalyptus globolous (Labill),
slightly inhibited the growth of S. aureus and E. coli but had
no effect on the growth or swarming of other strains. Lawsonia inermis L.,
at the concentration of 1% (v/v) had inhibitory effect on S. aureus,
one strain of P. vulgaris and one strain of P. penneri. The other
strains of P. vulgaris and P. penneri had very limited growth
(Fig. 1C). In this medium, one strain of P. mirabilis had
the typical swarming which covered nearly the entire plate. At a concentration
of 1.25% (v/v) of this extract only four bacterial strains had obvious growth
and one strain of P. penneri swarmed, although the colony size was small
(Fig. 1D). In a medium containing 2% (v/v) liquid extract
of Lawsonia inermis L., the growth of all bacteria except E. coli
was inhibited. In the presence of the essential oil of Glycerrihizia
glabra, (concentrations of 0.5 to 2% v/v) all the bacteria were able to
grow and swarming for all the strains were in a form of thick homogenous zone
of bacterial growth (Fig. 1E).
Methanolic extract of Zataria multiflora at different concentrations
had no effect either on growth or swarming of the isolates, whereas the essential
oil of Zataria multiflora was among the most active plant materials
tested. At the concentration of 1.25% (v/v), all the bacteria were able to grow
but only two strains of P. mirabilis and one strain of P. penneri
swarmed (Fig. 1F). When the concentration of this extract
was raised to 2% (v/v), the swarming for all of the strains were completely
inhibited. However at this concentration one strain of P. penneri, the
standard strain of P. mirabilis and S. aureus failed to grow.
Zataria multiflora at higher concentrations (2%) had growth inhibitory
effects for many bacterial strains tested. This essential oil (concentration
of 1.25 and 1.5% v/v) had slight effect on the swarming when the medium was
supplemented with 5-7.5% blood.
In presence of a mixture containing equal amount of essential oil of Lavandula officinalis, plus Ferula gumosa boiss, (Galbanium anti acne), although the bacterial growth was not inhibited, swarming was abolished and only one strain of P. penneri and one strain of P. mirabilis showed a very slight swarming. At a concentration of 2% (v/v) all the isolates were able to grow and there was no swarming. This medium preserved its anti-swarming activity when the medium was supplemented with blood and the hemolysin production on this medium was very clear such as the original blood agar plates.
||Effect of several plant extracts and essential oils on growth
of 12 different bacterial species and swarming behavior of Proteus mirabilis,
P. vulgaris and P. penneri. A) Essential oil of Gumium cymium
L., B) Essential oil of Rosa damascene; C) Fluid extract of
Lawsonia inermis L., D) Fluid extract of Lawsonia inermis L.,
E) Methanolic extract of Glycyrrhizia glabra L. and F) Essential
oil of Zataria multiflora Boiss, at concentration of (v/v) of 1.5,
1, 1.25, 2 and 1.25%, respectively. Plate abbreviations indicated are: M:
Protues mirabilis, V: Proteus vulgaris, PE: Proteus penneri,
E: Escherichia coli, ATCC 25922; S: Staphylococcus aureus,
ATCC 25923: MS Protues mirabilis, ATCC 43071 and P: Pseudomonas
aeruginosa ATCC 27853
Proteus rods had proteolytic and hemolytic activities, having considerable resistance to sodium chloride and are grouped among important putrefying bacteria[10,12]. P. mirabilis and P. vulgaris are also well known opportunistic pathogens, causing urinary tract infections and are also responsible for systemic and localized infections[3,6]. Several potential virulence factors may be responsible for the pathogenic properties of these bacteria[7,13]. It has been shown that swarming differentiation and expression of virulence factors, such as protease, hemolysin and urease, are co-ordinately regulated in P. mirabilis[5,8,13]. Recurrent urinary tract infections usually had a poly microbial origin and the identification of all the bacteria involved in the infection is important. Therefore swarming of Proteus in solid media used for the isolation of these bacteria may over whelm the growth of other pathogens thereby annulling plans for adequate antibiotic options.
Many reports show the effectiveness of traditional herbs against microorganisms[2,14,15]. Plants are indispensable source of chemical compounds and there are many interests to investigate natural material as a source of new antibacterial agents. The aim of this study was to find a natural non toxic compound that can promote the growth of pathogens but inhibit swarming.
It has been shown that swarming of different strains of proteus may
fail to penetrate into each other and a line of demarcation is produced between
the two swarmers (Dienes phenomenon). The interesting finding
in this study was the effect of plant materials on the pattern of swarming and
Dienes phenomenon. For example in the presence of essential oil of Gumium
cyminum L., swarm cells of P. mirabilis and P. penneri spread
outwards and formed the synchronous oscillating waves around the colonies (Fig.
1A). However in the medium containing the essential oil of Rosa damascene
L., swarming pattern was changed and a homogenous expanding zone of growth
was seen around the colonies (Fig. 1B). The place of demarcation
line could be also compared in Fig. 1A and 1B.
Since the production of this line is unrelated to the flagellar antigen of the
strain, but appears to depend both on the bacteriocin produced and the bacteriocin
to which the bacteria are sensitive, its possible that these plant
materials, increased or decreased the production of bacteriocin in some strains.
The most active plant materials tested were the essential oil of Zataria multiflora and a mixture containing equal amount of essential oil of Lavandula officinalis, plus Ferula gumosa Boiss, (Galbanium anti acne). The essential oil of Zataria multiflora had both antibacterial and anti-swarming activity, but its anti-swarming activity could not be applied to blood agar plates. Therefore the mixture containing essential oil of Lavandula officinalis and Ferula gumosa Boiss, has superiority to the essential oil of Zataria multiflora in respect to swarming, since it has no antibacterial activity and the anti-swarming property of this mixture is quite active in presence of blood, it can be applied to blood agar plates in order to support the growth of fastidious bacteria.
Effect of these plant materials on swarming and growth inhibition is not clear and further works are required to find the active constituent of plant material to clarify their mechanism of action. However since the non-swarmers are not virulent, these essential oils especially of Zataria multiflora which is generally used as an oral medicinal plant, can be used for food spoilage control, cosmetics and pharmaceutical use. Further studies will be required to elucidate the cell target of the essential oil components.
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