Molecular detection and antimicrobial resistance of Aeromonas from houseflies ( Musca domestica ) in Iran

Objective. This study aimed to report the molecular detection and antimicrobial resistance of Aeromonas among houseflies (Musca domestica) in Shahrekord and Isfahan provinces of Iran. Materials and methods. Flies were caught from household kitchens, cattle farms, animal hospitals, human hospitals, slaughter house and poultry farms and put in collection separate sterile tubes. Isolation was accomplished by culture of flies in alkaline peptone water followed by identification with Aeromonas-specific Polymerase Chain Reaction (PCR). Results. Out of 600 houseflies 73 (12.2%) were infected with Aeromonas spp. Significantly higher frequencies of Aeromonas were isolated in Shahrekord province (13.0%; 39/300) than in Isfahan province (11.3%; 34/300). The recovery frequencies of the organisms were significantly lower in kitchens as compared to those in cattle farms and hospital wards which were similar. Higher proportions of infected flies were obtained during summer whereas low proportions were obtained during winter. Conclusions. It is concluded that houseflies do harbor diarrheagenic pathogens, including Aeromonas especially during summer. The carried organisms are resistant to a number of antimicrobials at different levels. Thus, future plans aimed at stemming infections caused by these organisms should take flies into account. Control efforts of infections caused by this particular bacterium should therefore take into account Musca domestica.


INTRODUCTION
Aeromonas is a diarrheagenic bacterium responsible for a number of zoonotic infections in humans.It is commonly found in aquatic environment in temperate and subtropical countries causing opportunistic infections in both aquatic and terrestrial animals.Some species of Aeromonas are known to be associated with human diseases affect the gastrointestinal tract such as gastroenteritis and extraintestinal infections (1,2).Extra-intestinal infections caused by Aeromonas include empyema, urinary and biliary tract infections, bacteriaemia, peritonitis; and skin and soft-tissue infections (3)(4)(5).
House flies (Musca domestica) are known to be involved in the transmission of important bacterial agents causing human and animal infections (6,7).The flies, having close association with different forms of excreta and decaying organic matters (7), represent a substantial public health risk whenever they have access to human food.Their body anatomy, secretions they make and their feeding habits enable the flies to pick up and disseminate several pathogens (6,8).To that effect, a number of authors have detected bacterial pathogens, including Helicobacter pylori, Salmonella and Campylobacter from houseflies (8,9).Consequently this study was conducted with the aim of prevalence of Aeromonas in the houseflies as determined by both culture and PCR method caught from Shahrekord and Isfahan provinces of Iran.
Sample collection.This study was conducted on 600 houseflies collected from important sites in Isfahan and Shahrekord provinces such as household kitchens, cattle farms, animal hospitals, human hospitals, slaughter houses and poultry farms.The flies were collected either by manual capture or by using sticky traps.
At the animal hospitals, flies were captured from the around animals, around yard hospitals and near a dumpster in the parking lot.At the farms (cattle farms and poultry farms), flies were trapped on or around animals, their faeces and their feed.At the human hospitals, flies were captured from the around yard hospitals and near a dumpster in the parking lot and near public room.At the household kitchens, flies were captured from the kitchen, the public dining room, and near a dumpster in the parking lot.At the slaughter houses, flies were captured from the slaughter houses and near a dumpster in the parking lot.
Following capture the fly samples were transported to the laboratory of Biotechnology Research Center at Islamic Azad University using separate sterile tubes to prevent any contamination due to mixing of the samples.In the laboratory, flies were identified and killed by refrigeration in -20 o C cold chamber.They were then placed in 5 ml peptone water and left at room temperature for 5 hr before being processed.
Isolation of bacteria from samples.Bacteria were isolated from both the peptone water solutions that contained fly samples and from the fly guts by the method described by Nayduch et al (10).DNA extraction.Genomic DNA was extracted from all isolates using CinnaGen DNA extraction kit (Cinnagen, Tehran, Iran) according to the manufacturer's instructions.The extracted DNA was quantified by spectrophotometric measurement at a wavelength of 260 nm according to the method described by Sambrook and Russell (11).Extracted DNA samples were stored frozen at -20°C until used for molecular analysis using Polymerase Chain Reaction (PCR) at the Biotechnology Research Center.
Gradient, Eppendorf, Germany) under the following conditions: 2 min of initial denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for 15 seconds, annealing at 56°C for 15 seconds and extension at 72° for 20 seconds.This was followed by final extension at 72°C for 2 min and then the products maintained at 4°C until processed (10).The amplified products were analyzed in 1.5% agarose gel.Electrode buffer was TBE (Tris-base 10.8 g 89 mM, Boric acid 5.5 g 2 mM, EDTA (pH 8.0) 4 ml of 0.5 M EDTA (pH8.0)combined all components in sufficient H 2 O and stirred to dissolve).Gels were stained with ethidium bromide.Aliquots of 10 μl of PCR products were applied to the gel.Constant voltage of 80 for 20 min was used for products separation.After electrophoresis images were obtained in UVItec documentation systems (UK).

Statistical analysis.
Statistical analysis was carried out using SPSS statistical software version 17.0 (SPSS Inc.Chicago, IL, USA).Descriptive statistics were computed to determine frequencies of fly samples positive for Aeromonas and frequencies of resistance to different antibimicrobials.Chi square test was used to determine significance of the observed differences in proportions.

Detection of the organisms among the flies.
The overall recovery frequency of Aeromonas spp.from the captured houseflies (n=600) was 12.2%.The recovery frequencies of the organism in the specific provinces were 13.0% (39/300) for Shahrekord and 11.3% (34/300) for Isfahan.The observed difference in these proportions was not statistically significant.Table 1 shows the percentages of Aeromonas recovery from flies sampled in each of the six locations.The recovery frequencies were significantly higher in houseflies captured in animal hospitals and slaughter houses.The seasonal recovery frequencies of the organisms are shown in table 2. The frequencies were significantly higher in summer and low during autumn (p≤0.05).

RESULTADOS
Detección de los organismos en las moscas.La frecuencia de recuperación global de Aeromonas spp.en las moscas domésticas capturadas (n = 600) fue 12.2%.Las frecuencias de recuperación del organismo Antimicrobial resistance.Different proportions of Aeromonas spp.were found to be resistant to each of the antimicrobials tested (Table 3).Higher frequencies of resistance were observed for cefalothin, ampicillin, tetracycline and nalidixic acid.

DISCUSSION
The present study demonstrates occurrence of Aeromonas (12.2%) in houseflies in the different locations involved in fly sampling.It is likely that the flies picked the organisms from the surrounding environments which could be contaminated by either sick animals, insects, food, water and even human beings as a result of unhygienic practices.
It was noted in this study that houseflies derived from slaughter houses, cattle farms, poultry farms and animal hospitals were more frequently positive for Aeromonas spp.than those derived from household kitchens and human hospital wards; some of these differences being statistically significant.This could bring us to a suggestion that these different locations do differ in the extent of contamination with the bacterium.Household en las provincias específicas fueron 13.0% (39/300) para Shahrekord y el 11.3% (34/300) para Isfahán.
kitchens and human hospital wards could be less contaminated as they receive much attention in terms of hygiene due to the nature of the services they provide.On the other hand slaughter houses, cattle farms and poultry farms are always subjected to contamination with fecal matter which is a well known carrier of many bacterial pathogens.
Our study investigated and revealed carriage of bacterial pathogens on both the external body parts and in the guts of houseflies.Some other studies which also involved investigations on both the external and internal body parts of the flies found that all these parts carried the organisms.These findings indicate that the flies may act as both mechanical and biological vectors of bacterial pathogens (13).
Hospital acquired infections are among the challenges that the medical professionals face.Their frequencies remain unacceptably high and are associated with excess morbidity, mortality and increased healthcare costs (14).Aeromonas spp.are among the responsible bacterial organisms for such infections, the common manifestation being bacteriaemia; more so in immunocompromised individuals (15).Our results, which show occurrence of Aeromonas in houseflies in hospital environments, are suggestive of the possibility that the flies play role in perpetuation of some of these infections.The flies are likely to disseminate the bacteria they carry to patients and/or workers through contaminating food and/or water.
Houseflies (Musca domestica) are of common occurrence in livestock farms with varying n u m b e r s d e p e n d i n g o n s e a s o n .T h e i r contamination with potential human and animal pathogens has been reported earlier (6,8).In the current study the flies collected from cattle and poultry farms were found infected with Aeromonas.Our results and those obtained by other researchers elsewhere reveal exposure of the flies, in farm environments, to these and possibly other microorganisms.
Many infectious diseases in temperate countries display seasonality, exhibiting patterns associated with weather conditions (16)(17)(18)(19).Such diseases display seasonal peaks in summer and alternating with low background levels of infection during winter (20)(21)(22).Understanding these seasonal trends in infectious diseases is important for improving disease surveillance and analysis of the seasonal differences in the risks for contracting those diseases.In the present study the Nuestro estudio investigó y reveló el transporte de bacterias patógenas tanto en las partes externas del cuerpo, como en los intestinos de las moscas domésticas.Algunos otros estudios que también incluyeron investigaciones de las partes externas e internas del cuerpo de las moscas, encontraron que todas estas portan los organismos.Estos hallazgos indican que las moscas pueden actuar como vectores de transmisión mecánica y biológica de los patógenos bacterianos (13).
Publicaciones recientes han informado la aparición de resistencia antimicrobiana entre las especies de frequencies of detection of Aeromonas from flies were high during summer and low during autumn.These findings and those of earlier works suggest that climatic conditions may be influencing infections with these organisms.
Recent publications have reported occurrence of antimicrobials resistance among Aeromonas species isolated from different hosts (1,15).This was also evident in the present study in which isolates of were resistant to a number of antimicrobials though at varying levels.Unlike observations by authors of the two studies (1,15) who reported more than 90% susceptibility to cephalosporins, aminoglycosides, fluoroquinolones; in the present study susceptibility to these antibiotics stood at <70%.Although on the basis of in vitro studies fluoroquinolones as well as third and fourth generation cephalosporins are suggested to be the drugs of choice for treatment of severe Aeromonas bacteriaemia (15), medical practitioners still need to take into account the existing complexity of in vitroin vivo correlation in antimicrobial susceptibility profiles displayed by Aeromonas spp.
With the findings of this and other researches, houseflies may be considered as important vectors of bacteria causing gastrointestinal diseases including Aeromonas in the study area and possibly other parts of Iran.Control efforts of infections caused by this particular bacterium should therefore take into account Musca domestica.The carried organisms are resistant to a number of antimicrobials at different levels.Thus, future plans aimed at stemming infections caused by these organisms should take flies into account.

Competing interests
Authors declare that they have no competing interests.
Con los resultados de esta y otras investigaciones, las moscas domésticas se pueden considerarse como importantes vectores de las bacterias causantes de enfermedades gastrointestinales, incluyendo las Aeromonas en las áreas de estudio y posiblemente en otras partes de Irán.Los esfuerzos para el control de las infecciones causadas por esta bacteria particular deben, por lo tanto, tener en cuenta a la Musca domestica.Los organismos transmitidos son resistentes a diversos antimicrobianos a diferentes niveles.Por lo tanto, los planes que en el futuro se orienten para detener las infecciones causadas por estos organismos deberán tomar en cuenta a las moscas.

Table 2 .
Recovery frequencies of Aeromonas spp.from houseflies captured during different seasons in Shahrekord and Isfahan provinces of Iran.

Table 1 .
Recovery frequencies of Aeromonas spp.from houseflies captured at different locations in Shahrekord and Isfahan provinces of Iran.