J Arthropod-Borne Dis, March 2020, 14(1): 88–96 B Odetoyin et al.: Frequency and Antimicrobial … 88 http://jad.tums.ac.ir Published Online: March 31, 2020 Original Article Frequency and Antimicrobial Resistance Patterns of Bacterial Species Isolated from the Body Surface of the Housefly (Musca domestica) in Akure, Ondo State, Nigeria *Babatunde Odetoyin1; Babatunde Adeola2; Olarinde Olaniran1 1Department of Medical Microbiology and Parasitology, College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria 2Department of Microbiology, Federal University of Technology, Akure, Ondo State, Nigeria (Received 05 May 2017; accepted 12 Mar 2020) Abstract Background: The emergence and spread of antibiotic resistant bacteria has become a serious problem worldwide. Houseflies are potential carriers of pathogenic and resistant bacteria and could be contributing to the global spread of these strains in the environments.We investigated the prevalence and antimicrobial resistant profiles of bacteria isolated from houseflies in Akure. Methods: Twenty-five houseflies were captured by a sterile nylon net from the slaughterhouse, garbage dump, human house, hospital, and eatery from 9:00am to 1:00pm when the flies were active and transported immediately to the labor- atory in sterile containers for processing. Bacterial loads were enumerated by serial dilution and plating on nutrient agar and selective media. Bacteria species were isolated by conventional isolation technique. Antibiotic susceptibility test was determined by the Kirby-Bauer disc diffusion technique. Results: Sixty-seven bacterial species were isolated from 25 samples that were collected. The predominant bacterial species was Escherichia coli (n= 31, 45%), followed by Klebsiella pneumoniae (n= 17, 25%), Staphylococcus aureus (n= 11, 16%) and Pseudomonas aeruginosa (n= 3, 4.3%). The bacterial load of the samples ranged from 9.7×105CFU/mL to 1.65×106CFU/mL. The results revealed that all isolates of Pseudomonas aeruginosa, Salmonella spp, and Proteus mi- rabilis were resistant to streptomycin and cotrimoxazole, augmentin and amoxicillin respectively. None of the S. aureus iso- lates was resistant to cotrimoxazole, chloramphenicol, sparfloxacin, augmentin, and ofloxacin. All isolates were multi-drug resistant. Conclusion: House flies that were collected from the slaughterhouse, garbage dump, human house, hospital, and eatery may participate in the dispersal of pathogenic and resistant bacteria in the study environment. Keywords: Houseflies; Antimicrobial resistance; Pathogenic bacteria; Vector; Infection Introduction The emergence of antimicrobial resistance in bacteria has become a serious global prob- lem (1). A WHO report indicates that this prob- lem is contributing to the increase in the cost of diagnosing and treating resistant infections (2). Today, the main concern is that antimicro- bial production pipelines are drying up and very few available therapeutic options are effective for common infections. Antimicrobial resistant bacteria are usual- ly spread from person to person, or from the non-human sources in the environment. Avail- able data indicate that the house fly, a known cosmopolitan pest with a worldwide distribu- tion and commonly found in close association with human activities could also contribute to the global spread of pathogenic as well as re- sistant bacteria in the environments (3-5). House- flies are known to act as mechanical vectors of pathogenic bacteria like Vibrio cholerae, Esch- erichia coli, Salmonella and Shigella (6-8). They pick up the pathogens on their bodies with the aid of their hairy proboscis and feet and dis- seminate them by regurgitating vomits and depositing faecal droplets during the feeding process. These vectors have also been report- *Corresponding author: Dr Babatunde Odetoyin, E-mail: odetoyin@yahoo.com http://jad.tums.ac.ir/ mailto:odetoyin@yahoo.com J Arthropod-Borne Dis, March 2020, 14(1): 88–96 B Odetoyin et al.: Frequency and Antimicrobial … 89 http://jad.tums.ac.ir Published Online: March 31, 2020 ed to be carriers of multi-drug resistant bacte- ria in hospital environments, and they may par- ticipate in the spread of resistant as well as path- ogenic pathogens within hospitals (9). Adequate control of these vectors would allow a reduc- tion of the transmission of these pathogenic and resistant bacteria. In spite of the awareness of the dangers posed by these flies and their link with poor environmental sanitation, factors/ prac- tices such as indiscriminate refuse dumping and waste disposal, bad drainage systems coupled with improper handling of food still abound in Nigeria. Since pathogenic microorganisms are widespread in our environment, there is abun- dant opportunity for flies to become contami- nated and, in turn, contaminate the environ- ment. Hence, the aim of the study was to iso- late and characterize the bacterial pathogens on the external surfaces of houseflies and de- termine the antibiotic resistance patterns of the isolated organism to commonly used antimi- crobials. The data from this study will provide information on the dangers posed by these flies in the environment. Effective communi- cation of the information is expected to gener- ate apposite ideas for acceptable and worka- ble interventions to control the spread of re- sistant pathogens in our environment. Materials and Methods Study location, sample collection and pro- cessing The study was carried out in Akure, the cap- ital of Ondo state which is located in the South- western part of Nigeria. Akure lies on latitude 7o15՛North of the Equator and on longitude 5o15՛east of the Greenwich meridian (10). Twenty-five samples of houseflies were cap- tured by sterile nylon nets from five different locations. Flies were caught from the selected sites (slaughterhouse, garbage dump, human house, hospital, and eatery) during the period of study with sterile nets from 9:00am to 1:00 pm when the flies were active. The collected flies were placed in sterile vials and transport- ed to the laboratory for identification by an en- tomologist. All species other than Musca do- mestica were removed. After identification, 2ml of sterile normal saline solution was added to each vial that contained the fly and shaken vig- orously for one minute. The fly was removed from the saline and was checked for bacteria dis- lodged from the external surface of the fly (6). Bacterial counts and Isolation of microorgan- isms The diluents used for the samples were the sterile saline solution. One ml was taken from each sample using a sterile syringe and added to nine ml of sterile distilled water in the test tubes. This dilution process was repeated until the 4th dilution was obtained. From the seri- ally diluted samples, 1ml each of the 10-4 di- lutions of the housefly samples was taken asep- tically with the use of a sterile syringe, and pour- plated on the nutrient agar plates, and then in- cubated at 37 °C for 24 hours. The colonies on the plates were then counted and their morpho- logical features recorded (11). From the nutri- ent agar plate, sterile inoculating loop previ- ously flamed to red-hot and cooled was used to pick different colonies from all the isola- tion plates and then streaked on nutrient agar and incubated at 37 °C for 24 hours; the mor- phological features of the distinct colonies along the line of streak were observed and used to infer the type of organisms present on the iso- lation plates (12). The biochemical character- ization of isolates was carried out as described by Cheesbrough (13). Antibiotic sensitivity test The antimicrobial susceptibility patterns of the isolates were determined by the Kirby-Bau- er disc diffusion technique on Mueller-Hinton agar (CM0337) (Oxoid Ltd., Basingstoke, Hamp- shire, England). Antibiotics tested were aug- mentin (30µg), ofloxacin (10µg), chloramphen- icol (30µg), gentamicin (10µg), (10µg), spar- floxacin (10µg), amoxicillin (25µg), ciproflox- acin (10µg), streptomycin (30µg), pefloxacin (5µg) and cotrimoxazole (30µg) (Remel, USA). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2020, 14(1): 88–96 B Odetoyin et al.: Frequency and Antimicrobial … 90 http://jad.tums.ac.ir Published Online: March 31, 2020 The plates were incubated at 37 °C for 24 hours. The diameters of the zones of inhibition were measured with a ruler and interpreted accord- ing to the guidelines of the Clinical and La- boratory Standard Institute (CLSI) (14). Statistical Analysis Data were presented as frequencies and per- centages. Independent T-test and analysis of var- iance test of SPSS version 20 software pack- age (SPSS, Inc. Chicago, Illinois) were used to determine the significance of the data. All p- values were two-sided and a p-value that was less than or equal to 0.05 was considered to be statistically significant. Results Microbiological analysis The results of the microbiological analysis showed that total bacterial counts ranged from 9.68×105CFU/mL to 1.65×106CFU/mL. The highest load of bacteria was found in samples from hospital (1.65×106CFU/mL), followed by eatery (1.60×106CFU/mL), garbage dump (1.59X106CFU/mL) and human house (1.28×106 CFU/mL). There was no significant difference in the mean load of bacteria from the five sam- pled sites (F= 2.7836, p= 0.0547). However, the mean load of bacteria from hospital was sig- nificantly higher (1.276×106CFU/mL) than the mean load of bacteria from slaughterhouse (9.68 ×105CFU/mL) (t= -2.79503, p= 0.0233) (Table 1). Organisms isolated from different samples Sixty-seven bacterial species were isolat- ed from the external surfaces of 25 identified house flies. These were divided into six gene- ra comprising Klebsiella, Staphylococcus, Esch- erichia, Pseudomonas, Proteus, and Salmonel- la (Table 2). The commonest bacterial species identified was E. coli (n= 31, 46.3%), followed by Klebsiella pneumoniae (n= 17, 25.4%), Staphylococcus aureus (n= 11, 16.4%) and Pseudomonas aeruginosa (n= 3; 4.5%). Of all the different sites sampled, garbage dumps and human houses harboured the high- est number of bacteria species (n= 15). Esche- richia coli was the commonest bacterial spe- cies isolated in the slaughterhouse (n= 3), hu- man house (n= 8), eatery (n= 6) and garbage dump (n= 9). However, K. pneumoniae was the commonest bacterial species isolated from the hospital (n= 7). Baseline resistance rates of isolated bacteria All isolates (100%) of P. aeruginosa, Sal- monella spp, and Proteus spp were resistant to streptomycin, cotrimoxazole, augmentin, and amoxicillin. No isolate of S. aureus was re- sistant to chloramphenicol, sparfloxacin, aug- mentin, and ofloxacin. Isolates of E. coli were commonly resistant to augmentin (n= 28, 90.3%) and cotrimoxazole (n= 20, 64.5%). The Least resistance rates were exhibited by K. pneumoni- ae (n= 1, 5.9%), E. coli (n= 1, 3.2%), and S. au- reus (n= 1, 9.1%) to ciprofloxacin (Table 3). Multidrug resistance was defined as the re- sistance of isolates to at least one antibiotic in three or more classes of antibiotics. As shown in (Fig. 1), all isolates were multidrug-resistant, with 66.6% of isolates of P. aeruginosa, 45.4% of S. aureus, 33.3% of Salmonella spp., 19.3% of E. coli strains and 5.9% of K. pneumoniae resistant to three or more classes of antibiotics. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2020, 14(1): 88–96 B Odetoyin et al.: Frequency and Antimicrobial … 91 http://jad.tums.ac.ir Published Online: March 31, 2020 Table 1. Bacterial load of sample (CFU/mL) (104)a Colony Slaughterhouse Human House Hospital Eatery Garbage Dump Plate 1 86 84 102 184 206 Plate 2 102 184 158 146 178 Plate 3 56 98 196 96 164 Plate 4 84 128 204 206 142 Plate 5 156 144 166 168 108 Average 96.8b 127.6 165.2b 160 159.6 A= F= 2.7836, p= 0.054752; B= t= -2.79503, p= 0.0233 Table 2. Number of isolates from all sample locations Isolates Sample locations Slaughter house (n= 12) Human House (n= 15) Hospital (n= 14) Eatery (n= 11) Garbage Dump (15) Total (67) Klebsiella pneumoniae 0 (0) 4(26.7) 7(50) 2(18.2) 4(26.7) 17(25.4) Escherichia coli 6 (50) 8(53.3) 2(14.3) 6(54.5) 9(60) 31(46.3) Staphylococcus aureus 3(25) 3(20) 3(21.4) 0 (0) 2(13.3) 11(16.4) Pseudomonas aeruginosa 1(8.3) 0 (0) 2(14.3) 0 (0) 0 (0) 3(4.5) Proteus spp 2 (16.7) 0 (0) 0 (0) 0 (0) 0 (0) 2(3) Salmonella spp 0 (0) 0 (0) 0 (0) 3(27.3) 0 (0) 3(4.5) Table 3. Antimicrobial resistant pattern of identified bacteria in all sample locations Isolates Antimicrobial agents PER GEN AMO CPX S SXT CH SP AU OFX Staphylococcus aureus (n= 11) 2 (18.2) 3 (27.3) 7 (63.6) 1 (9.1) 3 (27.3) 5 (45.5) 0 (0) 0 (0) 0 (0) 0 (0) Salmonella spp (n= 3) 1 (33.3) 0 (0) 1 (33.3) 1 (33.3) 3 (100) 1 (33.3) 1 (33.3) 0 (0) 3 (100) 1 (33.3) Proteus spp (n= 2) 1 (50) 1 (50) 2 (100) 1 (50) 2 (100) 2 (100) 1 (50) 1 (50) 0 (0) 2 (100) Klebsiella pneumonia (n=17) 4 (23.5) 5 (29.4) 8 (47.1) 1 (5.9) 9 (52.9) 2 (11.8) 1 (5.9) 2 (11.8) 13 (76.4) 11 (64.7) Escherichia coli (n= 31) 2 (6.5) 3 (9.7) 18 (58.1) 1 (3.2) 23 (74.2) 20 (64.5) 4 (12.9) 2 (6.5) 28 (90.3) 5 (16.1) Pseudomonas aeruginosa (n= 3) 2 (66.7) 1 (33.3) 1 (33.3) 1 (33.3) 3 (100) 3 (100) 1 (33.3) 1 (33.3) 0 (0) 1 (33.3) PER= Perfloxacin (PER 30µg), GEN= Gentamycin (GEN 30µg), AMO= Amoxicillin (AMO 30µg), CPX= Ciprofloxacin (CPX), S= Streptomycin (S 30µg), SXT= Cotrimoxazole (SXT 30µg), CH= Chloramphenicol (CH 30µg), SP= Sparfloxacin (SP 10µg), AU= Augmentin (AU 30µg), and OFX= Ofloxacin (OFX 10µg) http://jad.tums.ac.ir Published Online: March 31, 2020 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2020, 14(1): 88–96 B Odetoyin et al.: Frequency and Antimicrobial … 92 http://jad.tums.ac.ir Published Online: March 31, 2020 Fig. 1. Multidrug Resistance patterns of isolates Discussion In the present study, we investigated the fre- quency and antimicrobial resistant profiles of bacteria isolated from houseflies in five differ- ent locations (slaughterhouse, garbage dump, human house, hospital, and eatery). Six bacte- rial species, comprising Salmonella, Proteus, S. aureus, P. aeruginosa, E. coli, and K. pneu- moniae were isolated from M. domestica. Sim- ilar findings have also been reported by previ- ous investigators who have highlighted the im- portance of houseflies as mechanical vectors of various pathogens (15-17). The external or- gans of M. domestica constitute a large source of bacteria, and their persistent association with humans, animals, food, refuse, and excreta makes them potential mechanical or biological vectors for the dissemination of pathogenic and multi- drug-resistant bacteria (18-22). The results of this study indicate that M. domestica plays a great role as a mechanical carrier of bacteria in this environment, most of the bacteria iso- lated which have also been isolated by previ- ous investigators are of medical importance (17, 23, 24). Of all the different sites sampled, garbage dump and human house harboured the highest number of bacterial species (n= 15). Garbage dumps are sites where waste products are kept, and they serve as media for breeding microbes. Also, these dumpsites serve as breeding sites for flies which while feeding could also convey microbes from one place to another thereby affecting the health of the community. Apart from garbage dumps, we also observed the high- est number of bacteria from human house and hospital which may be due to improper waste disposal and environmental sanitation. Our find- ing is in tandem with the study of Nazari et al. (25) that also reported in large numbers the iso- lation of bacteria from hospital and non-hospi- tal environments which they attributed to a low level of general hygiene. Escherichia coli was the commonest bac- terial sp isolated in the slaughterhouse (n= 3), http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2020, 14(1): 88–96 B Odetoyin et al.: Frequency and Antimicrobial … 93 http://jad.tums.ac.ir Published Online: March 31, 2020 human house (n= 8), eatery (n= 6) and garbage dump (n= 9). However, K. pneumoniae was the commonest bacteria sp isolated from the hos- pital (n= 7). Klebsiella spp and E. coli are gram- negative organisms that occupy a variety of niches like other members of enterobacteri- aceae. Their isolation from nearly all sources may not be unconnected with their ubiquity, and with the fact that house flies feed mainly on feaces and other animal waste, which is a rich source of enteric bacteria (26). Escherich- ia coli is known to cause diarrhoea including traveller's diarrhoea, and haemolytic uremic syn- drome, which people can contract by eating con- taminated food. Isolation of this pathogen from the eatery, the slaughterhouse, the human house could lead to outbreaks of E. coli gastroenteri- tis as these insects may deposit the pathogens when they feed on food that people consume (27). We observed a preponderance of isolates of Proteus from the slaughterhouse and iso- lates of Salmonella from the eatery. This ob- servation is in line with the findings of Urban and Broce (28) that reported Proteus spp. as the most common bacteria among Gram-neg- ative bacteria isolated from flies that were as- sociated with raw meat, followed by Providencia spp., Pseudomonas spp., and Salmonella spp. (29). Proteus species are well known as hu- man opportunistic pathogens and intestinal mi- croorganisms indicating fecal pollution of wa- ter or soil. Their isolation from flies from the slaughterhouse may be due to the use of such contaminated water for meat processing. In addition, Proteus spp. are able to produce vol- atile components such as putrescine and am- monia, which are important for their swarm- ing ability and, are also able to attract flies to animal carcasses (30). The isolation of Salmo- nella spp from eateries may portend grave danger to public health as this pathogen is associated with gastroenteritis. Of all the different bacterial species isolat- ed from flies in this study, S. aureus was the only gram-positive organism. This organism was isolated from nearly all the locations we sampled demonstrating its ubiquity. Our find- ing supports previous reports indicating that fly can carry S. aureus (25, 31). Staphylococ- cus aureus, an opportunist pathogen, is re- sponsible for a number of human diseases rang- ing from skin lesions, wound infections and food poisoning to more serious conditions, such as osteomyelitis, endocarditis and septicaemia (32). Therefore, its isolation from the human house and the slaughterhouse portends serious danger to public health. The results of antibiotic susceptibility tests showed that all isolates were multidrug-re- sistant. All isolates of P. aeruginosa, Salmo- nella spp, and Proteus spp were resistant to streptomycin, cotrimoxazole, augmentin, and amoxicillin. Isolates of E. coli were common- ly resistant to augmentin and cotrimoxazole. The least resistance rates were exhibited by K. pneumonia, E. coli and S. aureus to ciproflox- acin. Even though, there is a paucity of data on the occurrence of antimicrobial resistance in bacteria associated with flies in Nigeria, inde- pendent studies across the globe have never- theless emphasized the role of flies in the dis- semination of resistant bacteria. In a recent global review of the role of flies in the spread of antimicrobial resistance, Onwugambaa et al. (33) revealed that ‘filth flies’ are colonized with clinically relevant antimicrobial resistant bacteria, such as extended-spectrum beta-lac- tamase, carbapenemase-producing, and colistin- resistant bacteria. In Iran, Davari et al. (5). Iso- lated cephalexin, chloramphenicol, ampicillin, and tetracycline-resistant bacteria from flies, with resistance against the antibiotics above 32.5%. In a similar study conducted in China, multidrug-resistant enterococci, staphylococ- ci, E. coli, K. pneumoniae, P. aeruginosa and Aeromonas hydrophila were identified in flies that were collected beside poultry feeding op- erations (34, 35). Likewise, Wei et al. showed that antibiotic resistant bacteria can persist in the gut of house and green bottle flies. Animal http://jad.tums.ac.ir/ http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=Aeromonas+hydrophila J Arthropod-Borne Dis, March 2020, 14(1): 88–96 B Odetoyin et al.: Frequency and Antimicrobial … 94 http://jad.tums.ac.ir Published Online: March 31, 2020 remains, garbage, hospital waste, and sewage samples are potential sources of resistant bac- teria. Hence, flies that are exposed to these sources can easily pick up and disseminate re- sistant bacteria (36). Interestingly, some multidrug-resistant bac- teria particularly, Klebsiella, Pseudomonas, and S.aureus were isolated from hospital environ- ments. Therefore, hospitals houseflies may al- so participate more in the spread of antibiotic resistance in the environment. The occurrence of multidrug resistance in clinical isolates is a serious problem due to the waning number of antibiotics used to treat human infections (2, 37). Data from independent studies suggest a link between the antibiotic resistance of food of animal origin, the antibiotic resistance of clinical isolates, and community health (38, 39). Nevertheless, this link remains a contentious issue because of insufficient information on the ecology of antibiotic resistance and virulence genes in the environment (36, 37). Conclusion In conclusion, this present study indicates that house flies (M. domestica) are carriers of pathogenic bacteria that pose a possible health risk to communities. The isolated bacteria were resistant to various commonly used antibiotics. In view of the findings of this study, there is a need for public health education programmes and awareness to be given to the peasants, elites, and patients in the study environment and ep- idemiological surveillance of food vending joints, major water sources, and hospitals to prevent ingestion of contaminated food and water. Suitable steps should be taken to con- trol the flies and monitor the susceptibility pattern of the pathogens they carry. Further study is needed to determine the type and di- versity of all microorganisms spread by flies as well as the epidemiology of the resistant bac- teria they carry. Acknowledgements We thank Mrs Oluwabunmi Esther Yinka and Mr Ogunleye Festus Ajayi for their tech- nical support. The authors declare that there is no conflict of interest. References 1. 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