Isolation and Identification of Bacterial Isolates from House Flies in Sulaymaniya City

The objective of this study was to isolate and identify bacteria found in natural association with adults of Musca domestica.In this study adults of Musca dometica were collected from four sites in Sulamani city,Kudistan,Iraq to isolate and identify bacteria on the external surface of house flies . Bacteria from fly samples isolated using the conventional isolation technique were E.coli , Pseudomonas aeruginosa , Staphylococcus albus , Staphylococcus aureus , Salmonella sp. and Klebsiella sp. The results of current study confirm that the houseflies are much more than nuisance and they pose potentially serious health risks. Consequently, the population of houseflies has to be controlled


INTRODUCTION
he house fly, Musca domestica L. is well-known cosmopolitan pest. It has a worldwide distribution and is found throughout the country in close association with human activities. It receives the common name of house fly by virtue of being the most common fly found in and around houses in addition to being a nuisance pest [1] ., It is a vector of many pathogenesis carrier of over 100 different pathogenic organisms including organisms for diseases, typhoid ,cholera, bacillary dysentery, ,ophthalmia neonatorum and infantile diarrhea as well as parasitic worms [2,3] .
House flies Musca domestica are of medical and veterinary importance due to their capacity to act as mechanical vectors of microorganisms originating in animal manure and other decaying organic substrates [4]. How-ever, its classification as a "disease causing fly" as follows (1) its confirmed association with food-borne pathogenus Escherichia coli , Salmonella, and Shigella,(2)the fact that it is ecologically associated with humans (synanthropic ),(3) its association with domestic vironments(endophilic) ,(4) its equal attraction to excrements and human food sources, and (5) its municative behavior that allows the house fly to easily move from heavily contaminated to human populated areas [5].
Houseflies Musca domestica have been shown to be carriers of several species of bacteria; this is because of their close association with decaying organic matters, garbage and faeces [6] .The hairy proboscis and feet with glandular hairs and pads that secrete sticky material enable the flies to pick up the pathogens on to their bodies. In addition the regurgitation of vomits and deposit of faecal droplets during feeding process contribute to the flies' ability to spread the pathogens [7,8] .The common house fly ,is a medically-important insect worldwide [9,10,11] . House flies have been implicated as vectors or transporters of various human pathogenus, including Vibrio cholerae, Entrobacteriaceae pathogens, Staphylococcus aureus, and Pseudomonas spp. [5, 9, and 12]. Transmission takes place when the fly makes contact with people or their food. As many as 500000 microorganisms may swarm over its body and legs [13] Insects, such as houseflies, that develop in decaying organic material may transmit antibiotic-resistance bacteria from the manure of animals and other decaying organic substrates to residential setting. The habitats in which it develops (e.g., manure), its dependence on a live microbial community, its feeding mechanism(regurgitation),its attraction to human food, and its ability to fly long distance make this insect a very good candidate for dissemination of fecal bacteria, including human and animal pathogens[ 10, 19,20].
In the present study we collected house flies from residential area of four locations in Sulamani city and isolated human pathogenic bacteria from the external surfaces of this flies. Afterward, we determined the susceptibility of the bacterial strains to different antibiotics.

MATERIAL AND METHODS Collection of flies
The study was confined to the city of Sulamani. House -flies were collected in batches of 10 flies each from four different places (i.e. slaughter houses, garbage dumps, restaurant dumps, hospital dumps).Flies were caught from the selective sites during the period of study with sterilized nets and from9:00 a.m to 1:0 p.m., when the flies are active. The collected flies were placed in to sterile 150 ml container, and flies were transferred immediately to bacteriological laboratory.

BACTERIAL ISOLATION Culture Media
The Following media were used in culturing the bacteria, Cetrimide agar, Manitol salt agar, Chocolate agar, MacConky agar, EMB agar, API-20E system.

Isolation of bacteria from flies
Bacteria from fly sample collections were isolated using the normal isolation technique. The flies were anesthetized by keeping them at 4C o for 5 minute [21]. Each flies transferred individually with sterile tweezers to agar plate, flies were kept alive on plates where they moved over the surface while feeding, walking. After 1 hour at room temperature, the flies were removed and the plates were incubated at 37C o for 24 hours [6]. Bacterial colonies presenting morphological differences were picked and streak on new blood agar plate's .The cultures were then observed daily for growth and all bacteria colonies subculture on to corresponding media and further incubated until pure colonies were obtained [7]. The bacteria were identified to the genus level by colony morphology, texture and Gram staining and species level for some of them by using API-20E system. Antibiotic susceptibility test was performed and the following antibiotics were used in this study: Tetracycline, Erythromycin, Ciprofloxacin, Gentamycin, Ampicillin, Amoxicillin, Penicillin, Nalidixic acid, Ferodantin, Amikacin, Rifadin [10].

Results
From the 40 house flies collected from slaughter house, gabage dumps, hospital dumps, and restaurant dumps at Sulamani city, house flies were found to carry several species of bacteria on the external surface. This study showed that six species of bacteria were isolated from Musca domestica. The bacteria isolated from these sites as represented in Ttable (1).
The most frequent bacteria isolated from houseflies coming from all locations of the city were Pseudomonas, Staph. aureus, Staph. albus, and E-coli while the least present bacteria were Klebsiella sp. and Salmonella sp. (Table -1). A total 40 house flies collected from slaughter houses, garbage dumps, restaurant dumps , hospital dumps. In general, flies may transport agents of diseases on and within their bodies. In this work, only the exterior (external surface) transmission of microorganism was studied. As mechanical vectors, this fly transferred microorganism by means of their leg or/and mouth parts. This was also reported from many other authors [15]. All flies investigated transmitted more than microorganisms were identified and demonstrated inTable (1). Figures (1, 2), show the primary screening of isolates from flies on selective media. Antibiotic resistance were tested for six bacteria cultures obtained from house flies to determine their disc sensitivity or resistance to 11 commonly used antibiotics Table (2). Among all the isolates from slaughter house E.coli and Pseudomonas resistance to Tetracycline , Erythromycin , Gentamycine , Ampicilin , Amoxillin ,Penicillin , Nalidixic acid , Ferodantin ,and Rifadin ), and they were sensitive to Amikacin ,Ciprofloxin respectively, while Staph aures resistance to Erythromycine , Penicillin , Nalidixic acid , Ferodantin , Rifadin and sensitive to Tetracycline , Cipromycine , Gentamycine , Ampicillin , Amoxilline , Amikacin .
Aureus showed resistance to Erythromycine , Gentamycine, Ampicilline , Amoxilline , Penicilline , Ferodantin , Rifadin and sensitive to Tetracycline , Ciprofloxaxcin , Nalidixic acid , Amikacin). More ever the Klebsiella was resistance to the most antibiotics Erythromycine , Gentamycine , Ampicilline , Amoxilline , Penicilline , Ferodantin , Amikacin , Rifadin and sensitive to Tetracycline ,Ciprofloxacin , Nalidixic acid . All the bacteria that isolated from hospital dump were resistance to all antibiotics except E.coli sensitive only to Ciprofloxacin on other hand all isolated bacteria from restaurant dump show totally resistance to all antibiotics .

DISCUSSION
The biology and ecology of Musca domestica make it an ideal mechanical vector of human and animal pathogens. Garbage, cattle barns, poultry houses, slaughter houses, and hospitals are sites where house flies can reproduce [22]. This study showed that six species of bacteria were isolated from Musca domestica ( Table -1).Our results are in accordance with other reports which highlight the importance of house flies in caring various pathogens [23,24,25,26] .
Many scientists indicated that the external organs of Musca domestica legs, wings, and mouth parts) constituted a large source of bacteria they isolated [24,27]. The results of this study indicated that Musca domestica could play a great role as a mechanical carrier of bacteriain this study, most of the bacteria isolated were medically important, including E.coli, Staph. aureus, Staph. albus, Pseudeomonas aeruginosa , Klebseilla , and Salmonella. These finding agree with the results of [28,29] which presence E.coli,P. aeruginosa , K. pneumonia ,and Proteus mirabilis on the external surface of house fly collected from slaughterhouse , zoo, and hospitals .There are more studies which confirm the role of house flies on transmission of different

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bacteria as a world wide agent transmission of bacteria is using different insect species and different methods of transmission. [30] isolated Salmonella enteritids ,S. infantis and S . heidelberg from house flies over poultry houses . 18 species of enteropathogenic bacteria from different cyclorrhaphan flies were isolated in Malaysia [31] . The isolates of E.coli , Staphylococcus aureus , Pseudomonas spp., Proteus spp.,in Ahvazc city [29] . This confirm that the housefly's body can act as a mechanical vector either bacteria or many microorganisms. We examined the antibiotic susceptibility and resistance patterns of six bacteria obtained from the houseflies by using 11 commonly used antibiotics ( Table 2). The types of bacteria in general were resistance to all antibiotics. These finding are in agreement with the finding of other studies which show that the enter bacteria Salmonella, E. coli, Klebsiella , Shigella isolated from house flies collected in hospital were found to be resistant to significantly more of the commonly used antibiotics that were tested than the Enterobacteria isolated from the flies caught in the streets, and Pseudomonas isolates frequently showed resistance to multiple antibiotics [32] . Multiple resistances to antibiotics are common among P. aeruginosa isolated from different clinical sources in Iran [33] . Insects such as house flies Musca domestica L. that develop in decaying organic material may transmit antibiotic-resistant bacteria from manure of animals and other decaying organic substrates to residential setting [10, 19,20]. Multidrug resistance in clinical isolates has become a serious problem due to a progressive decline in the number of antibiotics that are effective for treatment of human infections [34]. It has been suggest that there is a connection between the antibiotic resistance of food animal origin, the antibiotic resistance of clinical isolates, and community health [35,36,37] . However, this remains a controversial issue [38,39] because the ecology of antibiotic resistance and virulence genes in the environment is not well understood. The present study indicates that the house fly Musca domestica poses a possible health risk to communities proved that the isolated strains of bacteria were resistance to various antibiotics. In conclusion, we report that house flies collected in slaughter house garbage dump, hospital dump, and restaurant dump may be involved in the spread of antibiotic resistant bacteria and may increase the potential for human exposure to antibiotic resistant bacteria. It recommended that suitable steps must be taken to control the flies.