Isolation, Characterization and Antagonistic Activity of the External Microflora of the House fly, Musca domestica (Diptera: Muscidae)

Experiments were designed to isolate, characterize and study the interaction between external microbiota (bacteria and fungi) carried by adult M. domestica after dipping, then removal of the flies from distilled water, sugar solution and saline solution. M. domestica was collected from Sakaka city, Northwestern Saudi Arabia. Three groups of adult M. domestica were completely dipped in and then removed from each of the above-mentioned solutions separately. Bacteria and fungi were isolated using corresponding media, characterized using macro and microscopic examinations, and then tested for antagonistic activity. Three bacterial species; Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa and three fungi; Candida albicans, Rhizopus stolonifer and Aspergillus niger have been isolated, characterized and tested for antagonism. Biochemical tests of bacterial strains confirmed the ability to secrete economically important materials. Different efficiencies to ferment sugars and produce gases have been confirmed, too. Antagonistic tests between microorganisms have revealed that both E. coli and P. aeruginosa bacteria are antagonists to both A. niger and C. albicans fungi. However, R. stolonifer fungus is antagonist to both E. coli and P. aeruginosa bacteria. B. subtilis bacterium is antagonist to the 3 fungi and to the other 2 bacteria. The antagonistic activity of our bacterial strains could be attributed to the secretion of antimicrobial materials. Further study on the mechanism of antimicrobial activity of B. subtilis strain is recommended. It was concluded that this strain could be useful in controlling some bacterial and fungal infections.


Collecting flies
The house fly, M. domestica, were collected from the Sakaka city, AlJouf, Northwestern Saudi Arabia. Collected flies were transported to the laboratory in sterile cups and then they were morphologically identified. M. domestica was reared and maintained in the insectary under controlled conditions (27±2 °C and 70±5% Relative humidity (RH) and 14/10 light/dark photoperiod cycle), according to 16 . These flies were used as a stock for the experimental work.

Solutions used
The experimental solutions were chosen to represent the normal drinks and foods of the human beings. Distilled water represents the normal drinking water of human. The 10% sterile sugar solution represents juices and other sugary drinks consumed by human. The 10% sterile saline solution represents the balanced salting of all types of salads and cooked foods with sauces. All solutions were sterilized using bacterial filters and all tools were autoclaved.

Experimental design
Three groups of adult M. domestica (10 flies/ group) were completely dipped in and then removed from each of the following solutions separately: 200 ml of sterilized distilled water (DW), 200 ml of 10% sterile sugar solution (SU), and 200 ml of 10% sterile saline solution (SA). Immediately after dipping and removal of flies, bacterial and fungal flora were cultured from the three solutions, separately (DW, SU and SA). One hour later after dipping and removal of flies, bacterial and fungal flora were cultured from the three solutions, separately (DW1, SU1 and SA1). Bacterial isolation using differential media A fixed volume (100 µl) of each of the solutions DW, DW1, SU, SU1, SA and SA1 was spread by sterilized scalpel on 20 cm diameter plates containing Nutrient agar (NA), Mannitol salt agar (MSA), MacConkey agar, Brilliant green agar (BGA) and Salmonella-Shigella agar (SSA) media, separately. Plates were sealed tightly with parafilm, placed upside down and incubated at 30 °C for 24-48 h. Plates were then investigated, bacteria were isolated, identified and stored until used in subsequent experiments. Procedure was carried out inside laminar air flow hood 17,18 .

Characterization of the Bacterial Isolates Phenotypic characterization
Phenotypic characterization of all isolates studied were performed and compared to phenotypic data of known organisms described in the Bergey's Manual of systematic Bacteriology 19 as well as Gram's staining according to the standard gram staining protocol 20 .

Antagonistic activity between bacterial isolates
Antagonistic activity was tested according to 21 . Briefly, 0.5 ml of a bacterial suspension was spread on the surface of solidified nutrient agar and paper-disc diffusion method 22 was used for the other bacterial strains. Clear inhibition zones were measured and compared to positive and negative controls. Each experiment was repeated thrice.

Fungal isolation
A fixed volume (100 µl) of the solutions DW, DW1, SU, SU1, SA and SA1 was spread onto 20 cm diameter plates containing Czapek-Dox's agar medium and Potato Dextrose Agar (PDA) medium, separately. Chloramphenicol (25.0 mg/ L) or Chlortetracycline (40.0 mg/ L) was added to the media to inhibit bacterial growth. Plates were sealed tightly with parafilm, placed upside down and incubated at 28 °C for 7-15 days 23 .

Identification of fungal isolates
Purification of the colonies was carried out by transferring each single colony to a sterile PDA plate and incubating plates at 28 °C for 7-15 days. The propagated colonies were mounted on slides and stained with lactophenol cotton blue to be examined under light microscope. Macroscopic morphology of mycelium and conidia was observed and used for fungal identification 24,25 .

Antagonism between fungi and associated bacteria
Antagonistic activity was tested according to (26). Briefly, one ml of each fungus was spread onto the surface of solidified Czapek-Dox's agar media. A paper-disc diffusion method was used as described above 22 . Three replicates were incubated at 30 °C for 15 days, and inhibition zones were measured and compared to a reference chart.

Characterization of bacterial strains
A total of 18 bacterial isolates were identified during this study from all samples. These isolates were isolated from DW, DW1, SU, SU1, SA and SA1. Isolates were definitely characterized as three species; Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa (Table 1).

Morphological characterization of bacterial colonies
Shapes, sizes, elevation, opacity and margins of the bacterial colonies are summarized in Table (2). All colonies were elevated and opaque except the translucent colony of E. coli. Circular colonies of E. coli and P. aeruginosa and irregular B. subtilis colony were observed, too. In addition, small-sized with entire margin colonies of E. coli, medium-sized with undulate margin colonies of P. aeruginosa and large-sized with lobate margin colonies of B. subtilis were noticed ( Table 2). Table (3) summarizes Gram's staining and cell morphology of the bacterial species. All bacterial cells were Gram-negative except B. subtilis which was Gram-positive. Meanwile, all cells were rod-shaped except P. aeruginosa which were coccobacilli.

Biochemical characterization of bacterial species
Specific biochemical assays were carried out to evaluate economic and commercial values of the species. All bacterial species secrete catalase, B. subtilis and P. aeruginosa secrete oxidase and only B. subtilis secretes urease (Table  4). These enzymes can be commercially harnessed and marketed.
IMViC tests indicated that only E. coli secretes tryptophanase enzyme and indole. Additionally, E. coli is glucose-acidic-fermenter.  (Table 4). In addition, TSI and H 2 S tests revealed that B. subtilis is trisugar-acidic-fermenter lacking both CO 2 and H 2 S gas production. E. coli is trisugaracidic-fermenter producing CO 2 and lacking H 2 S gas production. Whilst, P. aeruginosa is non-trisugarfermenter (Table 4).

Characterization by differential media
In order to differentiate between the obtained bacterial species, 5 differential media were employed. Bacterial growth and characteristic colors of bacterial colonies were summarized in Table (5). Three growths with two characteristic colors were observed with MacConkey agar, two growths with two characteristic colors with NA media, only one growth with a characteristic color was observed with SSA, BGA and MSA media ( Table  5). Insufficient characterization has been observed when using differential media.

Antagonistic activity between bacterial species
Growth of two or more microorganisms in a single culture medium may indicate synergistic activity. However, growth of a single species on the medium may indicate antagonistic activity of the growing species. Our results revealed that B. subtilis is antagonistic to both E. coli and P. aeruginosa (Table 6).

Fungal isolation
A total of ten fungal isolates were isolated during the current work. Only one isolate from DW and DW1, two isolates from SU and SU1, two isolates from SA and SA1 were isolated. Fungal isolates were identified as Candida albicans, Rhizopus stolonifer and Aspergillus niger ( Table  7). C. albicans was persistent in all solutions, R. stolonifer appeared in sugar solutions and A. niger grew in salt solutions ( Table 7).

Characterization of fungal isolates Macroscopic and microscopic characterization
Table (7) clarified that all fungal isolates were identified to three different species; C. albicans was isolated from all solutions (6 isolates), R. stolonifer was isolated from sugar solutions (2 isolates) and A. niger was isolated from salt solutions (2 isolates).  (Table 8).

Antagonistic activity
E. coli and P. aeruginosa bacteria prohibited growths of both A. niger and C. albicans, whatever bacteria have applied individually or in combination. However, R. stolonifer prohibited growths of E. coli and P. aeruginosa whatever applied to the fungus individually or mixed with each other. Interestingly, B. subtilis bacteria prohibited the growths of all fungi whatever it has applied individually or in combination with other bacteria (Table 9).
The antagonistic activity of our bacterial strains could be interpreted by the ability of bacteria to secrete enzymes and other economic materials as shown in biochemical characterization. Antagonistic tests between microorganisms have revealed that both E. coli and P. aeruginosa bacteria are antagonists to A. niger and C. albicans fungi. Agreeable results have been presented by 51 who revealed that E. coli secretes a fungicide that kills C. albicans. Also P. aeruginosa was reported as antagonist to A. niger 53 . Other studies have reported that P. aeruginosa is antagonist to Aspergillus fumigatus in planktonic growth 54 and in bio lm, too [55][56][57][58] . Contrary to our results, no antagonism between E. coli and C. albicans has been found 26 . Interestingly, P. aeruginosa and A. fumigatus have been reported to possess mutual antagonism at different stages of bio lm development 59 . Recently, the complexity beyond the simple antagonistic interaction between P. aeruginosa and C. albicans has been intensively reviewed 60 . E. coli, Pseudomonas sp. and Bacillus sp. have been reported as antagonists to A. niger and could be used in biocontrol of the fungus 61 . E. coli has exhibited antagonistic activity to pathogenic Aspergillus spp. 62 . However, R. stolonifer fungus is antagonist to E. coli and P. aeruginosa bacteria. A previous study has presented that R. stolonifer fungus showed antagonistic effect to A. niger and C. albicans fungi and to P. aeruginosa and E. coli bacteria. This activity was attributed to toxic secondary metabolites secreted by the fungus 63 . B. subtilis bacterium is antagonist to the 3 fungi and to the other 2 bacteria. In antagonistic study, B. subtilis has proved to produce a biosurfactant that prohibited the growth of Salmonella, Shigella and Staphylococcus bacteria 64 . Antifungal activity of Bacillus isolates against phytopathogenic fungi may be attributed to the cyclic lipopeptide; fungycin which plays important role in this process [65][66][67][68] . Recently, the antimicrobial compounds of B. subtilis have been intensively reviewed 69 . No microbial competition between bacteria and fungi was recorded in the present study. However, microbial competition after natural falling and dipping of house fly in water and milk has been reported 15 . The total number of microbes has decreased within one hour after dipping in the case of water. Meanwhile, immediate decrease in total number of microbes in the case of milk has been reported 15 . Further research on the effect of falling and dipping of M. domestica using electron microscopy and molecular techniques is recommended.
Overall, the current work presents isolation, characterization and antagonistic activity of six microorganisms isolated from external surface of the house fly; M. domestica after dipping in DW, SU and SA solutions. Our results revealed that our bacterial strains secrete many economically important materials which could be harnessed and marketed. Different efficiencies of sugar fermentation and gas production have been observed, too. In addition the antagonistic activity, especially the ability of B. subtilis bacterium to prohibit growth of all bacterial and fungal strains could be interpreted in the light of its production of bioactive materials. Further study on the mechanism of antimicrobial activity of B. subtilis strain is recommended. We concluded that this strain could be useful in controlling some bacterial and fungal infections.

ACKNoWlEDGEMENTS
We thank Dr. Shaymaa Nabil for her technical support.

CONFliCts OF iNteRest
The authors declares that there is no conflict of interest.

AUTHorS' CoNTrIBUTIoN
Conceived and designed the experiments: FHG, AMS. Performed the experiments: FHG, AMS, TES. Analyzed the data: FHG, AMS. Wrote the paper: AMS, FHG. All authors have approved the final manuscript.