Housefly ( Musca domestica ) as Carrier of Enterotoxigenic Staphylococcus aureus in Broiler Farms in Iran : Is it Important for Public Health ?

Background: During the last decade, the prevalence of insect borne diseases due to contaminated food as well as the outbreaks of diseases due to enterotoxin-producing Staphylococcus aureus (ETSA) strains has increased. Objectives: This study was conducted to determine the prevalence rate, enterotoxigenecity, and antimicrobial resistance of S. aureus isolated from M. domestica collected from the residential areas of nine districts broiler farms in Zabol, Iran. Materials and Methods: The flies were captured with a sterile nylon net and washed twice with distilled water. The contents were streaked onto selective media and S. aureus was identified using the conventional biochemical tests. Antimicrobial susceptibility testing was performed using CLSI guideline. Staphylococcal enterotoxins (SEs), SEA, SEB, SEC and SED, was detected by the reverse passive latex agglutination (PRLA) method. Coagulase typing was performed with coagulase typing kit. Results: A total of 87 (17%) S. aureus was isolated from 450 samples. The ability to synthesize staphylococcal enterotoxins (SEs) was determined in 62 of 87 (71%) isolates. SE type B was the most common enterotoxin found in the isolated S. aureus (45%), followed by SE type A (26%), SE type C (5%), SE type D (8%), SEA + SEC (7%) and SEA + SEB (7%). Among the antibiotic tested, Penicillin was the most resistant antibiotic tested. Sixty seven percent of isolates belonged to coagulase type III, VI, VII, VIII, IV, and V. Conclusions: Our results suggest that S. aureus, including ETSA, is being carried by house flies such as M. domestica and may contribute to the spread of pathogenic isolates, with an impact on public health.


Background
The housefly, M. domestica, is important to public health since it acts as a potential vector for a range of various pathogenic bacteria present in the human and domestic animals (1,2).Domestic flies have evolved to live in proximity to humans and have been found to carry a number of etiological agents of typhoid and paratyphoid fevers, bacillary dysentery, cholera, hematic carbuncles, bovine mastitis (garget), conjunctivitis, and poliomyelitis (3).Houseflies greatly enhance the risk of human exposure to a wide variety of foodborne pathogens due to their uncontrolled movements, ability to fly short or long distances, and attraction to raw, processed and cooked food material (4).Houseflies can carry human pathogens from broiler farms, hospitals, public parks, garbage/ dump areas, slaughter Houses (5), and domestic habitation (6).
The houseflies have potential to transmit pathogens such as E. coli and S. aureus, fungi, and helminthes (7,8).It has been demonstrated that some of these microorganisms may live inside and/or on the housefly body surface from 5 -6 hours up to 35 days (9,10).Among the pathogens commonly transmitted by house flies include pathogenic bacteria that cause illness and food poisoning in humans.
Staphylococcal food poisoning caused by enterotoxin producing S. aureus is an important food born disease throughout the world.Dirt, soil, body discharges, and excreta from animals in holding pens are the main sources of staphylococcal contamination of house flies (8,11).Escherichia coli and S. aureus are commonly isolated from the indoor environments, including hospitals (12,13).

Objectives
The present study was conducted to isolate and identify ETSA of medical importance from house flies which were collected from the broiler farm environments.

Materials and Methods
The flies were captured with a sterile nylon net (5) and transferred to the microbiology laboratory at the faculty of veterinary medicine, placed in a sterile dish and frozen at -20 o C for 15 min to anaesthetize them.Twenty five flies were placed individually in test tubes containing 5 mL PBS 1% and another 25 flies were washed twice with distilled water and dissected with sterile micro dissecting needle and the gut contents of each individual fly was placed in 1.5 mL of 1% sterile PBS

Isolation of Bacteria From Bamples
The contents were streaked onto mannitol salt agar (Hi-Media, India), nutrient agar (Hi-Media, India), and 5% sheep blood agar (Hi-Media, India), and were incubated at 37°C for 24 hours.After incubation, colonies exhibiting morphology resembling staphylococci characteristics were selected, purified and identified further using the conventional tests, such as Gram staining, production of catalase, oxidase, coagulase, phosphatase, staphylokinase, DNAase, and fermentation of sugars (13,14).

Enterotoxin Analysis
The "SET -RPLA" kit (Oxoid, Basingstoke, UK) that relied on rapid reverse agglutination to detect SEA, SEB, SEC, SED in S. aureus was used.All of isolates of S. aureus were tested and results were interpreted in accordance with manufacturer's instructions.

Biotyping
Biotyping was carried out according to simplified system established by Devriese et al. ( 16) which uses four differential diagnostic tests: screening for staphylokinase and β-haemolysin production, coagulation of bovine plasma within 6 h, and the type of growth on crystal violet agar (16,17).

Coagulase Typing
Coagulase typing was performed using a coagulase type-specific antiserum (types I to VIII) kit (Denka Seiken, Japan).Briefly, 0.1 mL of each antiserum was mixed with normal rabbit serum (as a control) and 0.1 mL of the supernatant obtained from the overnight culture of each S. aureus isolate and incubated at 37°C for 1 hour.Subsequently, 0.2 mL of rabbit plasma was added to each tested tube, followed by incubation at 37°C for at least 1 hour.Inhibition of the coagulation indicated the coagulase type, which can easily be confirmed by simply decanting the contents of the tube.A strain is designated as being non type able (NT) if none of the antisera (types I to VIII) are able to neutralize the coagulase activity

Discussion
House flies have been considered transmitters and spreaders of pathogenic bacteria in hospitals and households or residential areas (18).The biology and ecology of M. domestica make it an ideal mechanical vector of human and animal pathogens (19,20).Cattle barns, poultry houses, slaughter houses and hospitals are sites where house flies can reproduce (21).Many scientists indicated that the external organs of M. domestica (legs, wings, and mouthparts) and internal organs constituted a large source of bacteria they isolated.The results of this study indicated that M. domestica could play a great role as a mechanical carrier of ETSA.These findings agree with the results of Barreiro et al. (22) in Portugal, which showed presence of Enterobacteriaceae, Escherichia coli, ETSA, and Listeria monocytogenes collected from kitchens, and kitchens situated in the closest proximity to the animal husbandry.Our results are in accordance with other reports which highlight the importance of houseflies in carrying various pathogenic bacteria, suggesting that these insects may serve as an important avenue of disseminating disease-causing agents like ETSA to the environment (9,23,24).ETSA were isolated from 14 % of M. domestica that sampled from the nine sites of broiler farms.Flies captured in the broiler farms showed a significantly higher presence of ETSA (62 out of 87 isolates).As we can see, there is a significantly larger prevalence of ETSA (70%) in the flies captured on the collection sites.Although there are not so many similar studies, these results are in agreement with those described in the literature (25).Our results suggested that houseflies in poultry farms play a role in the dissemination of ETSA among poultry and communities living in close proximity to animal operations that harbor heavy fly population.In the present study 70% of the isolates examined were enterotoxigenic.SEB was most frequently found in ETSA isolates and the next frequency was SEA.Among the wide range of staphylococcal virulence factors, SEA is one of the most common toxin have been clearly involved in staphylococcal food poisoning, and it is also associated with several other serious medical illnesses.Therefore, the presence of potentially life threatening pathogens on the captured flies showed that poses a high risk to the health of individuals, especially if we consider its role in staphylococcal food poisoning.In the present study, S. aureus isolates were examined for their production of only four classical SEs by SET -RPLA assay.Further work is needed to determine the presence of these new SE genes.In present study, 26% (23 out of 87 isolates) isolates were coagulase type VII, followed by VIII (18%) and IV (8%) that is in agreement with the other study (26).Most of the S. aureus isolates were resistant to ampicillin and ampicillin.In this study, Vancomycin, ciprofloxacin and ofloxacin were the most effective antibiotics against the studied isolates.Uncontrolled uses of antimicrobial drugs in medicine and food animal production accelerate the emergence of drug-resistant strains in human and livestock.Use of antimicrobial agents in poultry industry can results in generation of antimicrobial resistance and therefore the bacteria excreted in their feces are likely to be potentially resistant to those agents.Flies breed and develop in close proximity with these broilers and are often present in the poultry litter and have unrestricted movement.The majority of the enterotoxin-producers showed a large population with antimicrobial resistance that can be carried by the flies into the human habitat.Graham et al. (25) reported that flies collected near confined poultry feeding operations may be contributed in the spread of drug resistant bacteria from these broiler chicken farms and may increase the potential for human exposure to drug-resistant bacteria.The same conclusion was reported by Davari et al. (27) in houseflies collected in hospitals and slaughterhouses, indirectly implicating house flies as a potential source of the contamination.
The emergence of enterotoxin-producers isolates with multiple antibiotic-resistant phenotypes is considered a serious health concern.Further, the carriage of antibiotic resistant bacteria by flies in environment increases the potential for human exposure to drug-resistant bacteria.

Figure 1 .
Figure 1.Coagulase Type Pattern of S. aureus Isolates.Coagulase Typing Was Performed Using a Coagulase Type -Specific Antiserum (Types I to VIII) Kit (Denka Seiken, Japan)

Table 1 .
Distribution of the SEs Among the 62 Enterotoxigenic Isolates of S. aureus Tested a Data are presented as (%).

Table 2 .
The Biovars of S. aureus According to Simplified System a a A = human biotype; C = animal biotype.

Table 3 .
Results of the Antimicrobial Susceptibility for the Tested S. aureus Isolates a a Data are presented as No. (%).