ISOLATION AND PARTIAL CHARACTERIZATION OF SALMONELLA PHAGE FROM MAROJARM PLANTS

The marjoram plant is recorded as medicinal one, traditionally recommended as stimulant / tonic , and help to reduce the severity of asthma, indigestion, headache, rheumatism and toothache. Results of this study showed high total count of contaminating bacteria in marjoram plants in non-packed samples collected from eight Egyptian governorates as compared with the pre-packed ones from two local companies. The presence of Bacillus sp. and E. coli, but not Salmonella sp. are note-worthy. One phage successfully infected Salmonella typhimurium was isolated using plaque assay which produced circular turbid plaques .The electron micrograph of negatively stained preparation indicated that the phage particles have hexagonal head (~80 nm) with short tail (~20 nm), which is classified to family Podoviridae. The biological characterization of the phage isolate is found to be : inactivation point at 62°C for 10 min., longevity in vitro for 72 hrs, dilution end point of 10 -3 and stability for 1.5 hrs at pH 7.2.


INTRODUCTION
Marjoram plants (Marjorena hortensis L.) and marjoram-related microbial populations with their corresponding phages should have received more research in Egypt.
In Egypt, the plant has a cultivated area of nearly 4000 feddan, mostly in the governorates of Giza, Fayum, and Menia (Ministry of Agriculture, 2005).It is traditionally recommended as stimulant / tonic , and help to reduce the severity of asthma, indigestion, headache, rheumatism and toothache.Purdue University (1997) reported some antifungal and anticancer activities.Egyptian researches on isolation of phages from medicinal plants are non-existent.The present study aimed to fill that gap.

Source of plant samples
From local shopes in eight governorates (five samples per each), non-packed marjoram plants were taken in consideration.Additionally, two local companies offered pre-packed samples (five per each) to be included in the study.All samples were maintained in plastic bags at 2-5°C until analyses.

Bacterial load
Steps for the total count of bacteria and yeast and the presence / absence of Bacillus sp., E. coli and Salmonella sp. were carried-out according to procedures detailed in the following references: -Total count of:

Source of bacterial isolates
Ten isolates of the following bacteria from the Virology Lab of Department of Microbiology , Ain Shams University Faculty of Agriculture are provided: Bacillus subtilis Bacillus megatherium

Sources of phages
The phage was isolated from the plant samples as recorded in number (1).

Detection and isolation of phages
A lytic phage was assayed qualitatively (spot test) and quantitatively (plaque assay) according to Robert et al. (1970).The single plaque isolation technique was applied to produce the pure lysate phage.

Determination of biological characters of the isolated phage
Tests / assays were carried-out to determine the biological characters of the isolated phage.The following references were used table (3 -Longevity in vitro Stringer (1986) -Stability at different pH levels Stringer (1986) The phage morphology was described using a transmission electron microscope (Type Jeol-Jem 1010, Regional Centre of Fungi, Al-Azhar University, Cairo , Egypt ) used negative staining technique utilizing phosphotengestic acid (PTA) at PH 6.8.showed that in marjoram the bacterial load is much higher than that of yeast.

RESULTS AND DISCUSSION
According to Table (2), samples from the companies did not exhibit any sign of Salmonella presence.However, they showed mild presence of Bacillus sp. and E. coli.Shops, on the other hand, gave six out of 8 samples free from Salmonella, together with mild presence of Bacillus sp. and extensive presence of E. coli.
From one sample (taken from BeniSuef governorate), a lytic phage was isolated and characterized as specific for Salmonella typhimurium (Fig. 1).After incubation, the isolate produced turbid and circular single plaques (Fig. 2).The isolated of Salmonella phage shows signs of remarkable stability in terms of thermal inactivation point of 62°C/10 min., longevity in vitro for 72 hrs., dilution end point of 10 -3 , stability for 1.5 hrs at pH 7.2.These results are in general agreement with those reported previously by Robert et al. 1970, andStringer, 1980.According to transmission electron micrographs (Fig. 3), the isolated Salmonella phage appeared to be of hexagonal head (~80nm) and short tail (~20 nm), indicating that it belongs to family Podoviridae as pointed out by In light of the present results it is safe to use packed marjoram plant due to the absence of salmonella sp.In case of using cold non-packed of marjoram plant, it is suggested to use salmonella phage as biological control after sequencing the genomes of lytic bacteriophages e.g: Salmonella phage vil for safety prior to food applications (Anany et al. 2011).
Sources of contamination of medicinal plants are multiple, among which are practices applied during harvesting, handling, drying, packing and storage.Kneifel and Berger (1974) emphasized the importance of drying and storage as potential sources of contamination.Specifically, contamination with Salmonella is mostly due to polluted surface / standing water (Tindall et al ., 2005 and Ryan & Ray, 2004).

Fig . ( 2
Fig .(2): plaque assay technique show plaques resulting from the of the lytic Salmonella phage.

Table ( 1
): Load of yeast and bacteria in samples of marjoram plants, non-packed (from * found in 10 g of dry leaf plant sample diluted in 90 ml sterile saline solution.

Table (
2): Presence / absence of Bacillus sp., E. coli and Salmonella sp. in samples of marjoram plants, non-packed (from local shops) and pre-packed (from local companies).