84-431-2-SP - Iranian Journal of Health, Safety and Environment

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Alan Davies
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Bacteria of Phlebotominae Sand Flies Collected in Western Iran
Somayeh Rafatbakhsh-Iran1, Aref Salehzadeh1, Rasoul Yousefimashouf2 ,
mohammad najafimosleh2, Zahra Karimitabar2, and Maryam khedri3.
1. Department of Medical Entomology and Vector Control, Faculty of
Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.
2. Department of Medical Bacteriology, Faculty of Medicine, Hamadan
University of Medical Sciences, Hamadan, Iran.
3. Department of Medical Parazitology, Faculty of Medicine, Hamadan
University of Medical Sciences, Hamadan, Iran.
Mobile: 09183122067
E-mail: [email protected]
Bacteria of Phlebotominae Sand Flies Collected in Western Iran
Background: Microorganisms particularly bacteria presenting in insects
such as Phlebotominae may have an important role in the epidemiology of
human infectious disease. Nowadays, because of vector implications, the
routine methods of controlling and spraying has no more useful effects on
vectors and reservoirs. Little is known about the prevalence and diversity of
sand fly bacteria.
This information is important for development of vector control strategies.
Methods: The microbial flora of Phlebotomus papatasi and P. sergenti the
main vector of Cutaneous Leishmaniasis in the old world, was investigated.
Bacterial strains were identified by routine microbiological methods.
Results: We characterized 8 bacteria isolates from Phlebotominae sand
flies, including 5 Gram-negative bacteria: Acinetobacter lwoffii,
Pseudomonas aeruginosa, Enterobacter cloacae, Edvardsiela sp. and
Proteus mirabilis and Gram-positive bacteria: Bacillus subtilis,
Staphylococcus saprophyticus and Micrococcus luteus.
Conclusion: Our study provides some data on the microbiota diversity of
field-collected sand flies for the first time in Hamadan, west of Iran. Our
results indicate that there is a range of variation of aerobic bacteria
inhabiting sand fly, which possibly reflect the ecological condition of the
habitat where the fly breeds. Microbiota are increasingly seen as an
important factor for modulating vector competence in insect vectors. So,
mirobiota can affects on the biology of phlebotominae and their roles in the
sandfly-Leishmania interaction.
Further experiments are required to clearly delineate the vectorial role of
sand flies. Because it is probable that in the future, factors such as
environmental changes, migration and urbanization can ease the
transmission of leishmaniasis in this area.
Phlebotominae sand flies (Diptera: Psychodidae) are important vectors of
leishmaniasis, Carrion's disease or bartonellosis, and a variety of arboviral
diseases (1-3). Not only are novel viruses currently being discovered in sand
flies, but also different reservoirs are being identified for pathogens and
parasites of human diseases, transmitted by sand flies. The distribution areas
of sand flies and the diseases they transmit are also expanding. New viral
diseases of humans transmitted by sand flies are being reported as well (47).
The disease can present in three main ways as: cutaneous, mucocutaneous,
or visceral leishmaniasis (8). Cutaneous leishmaniasis is more prevalent
throughout the world and causes disfiguration and other associated
complications. Anthroponotic Cutaneous leishmaniasis and Zoonotic
cutaneous leishmaniasis caused by L. tropica and L. major, respectively, are
widely distributed in Turkey, Egypt, Israel, Iran, Saudi Arabia and the
northern part of India, where mainly P. sergenti and P. papatasi have been
incriminated as the vectors (9).
The disease is endemic in many rural districts in 17 out of 31 provinces of
Iran (10). In Hamadan Zahirnia and et al. carried out an epidemiological
survey in Hamadan that indicated occurrence of about 210 cases of
cutaneous leishmaniasis in this province. According to their study 99% of
the patients had a history of traveling to staying at endemic areas (11).
One of the most important factors in transmission of leishmaniasis is the
presence of sandflies harboring leishmanial infection (12).
Adult sand flies usually remain close to their larval development sites (13).
The sites where larval development take place are usually a mixture of
animal faeces and mud which are found in both wild and anthropized
biotopes (14). Larvae feed on the decomposing organic materials in these
sites and the adults can therefore acquire a part of their microflora during
their larval development. Furthermore, male and female sand flies feed daily
on natural sugars, especially nectars or sap secretions and drink water from
plants (15). These sugars are the main source of carbohydrates for adults.
Additionally, females require a blood-meal to complement their diet, during
the maturation of their eggs and completion of the gonotrophic cycle (16).
During these feeding events, they can also acquire various microorganisms
including bacteria (e.g. Bartonella bacilliformis), fungi, Phleboviruses or
other trypanosomatidae and co-colonization by human pathogenic and non
pathogenic species of Leishmania (17-18).
In addition such bacteria may interfere with the development of medically
important pathogens. For example, high midgut counts of Gram-negative
bacteria are known to significantly reduce oocyst numbers in plasmodiuminfected mosquitoes (19). Clearly, a complex interaction exists between
Leishmania and midgut microbiota that has an effect on the development of
mature infections (20).
Based on few previous studies it is believed that the microorganisms
existing naturally in wild and laboratory-reared insects might have an
important role as determinants of parasite survival and development in
insect hosts. Some bacteria have attracted attention because they induces a
number of intriguing abnormalities in the host’s reproductive system (21).
Also, intracellular microorganisms affect the biology of their invertebrate
hosts in many ways, ranging from mutualistic effects to the establishment of
reproductive isolation and speciation (22-23). A very preliminary study on
P. papatasi from Morocco identified just two bacteria (24).There is also a
small report on the distribution of bacteria from P. papatasi collected in
Egypt (25). Adler and Theodor suggested as early as 1929 that the presence
of microbes in sand flies might interfere with Leishmania infection (26).
Later, Schlein et al. saw a reduction of infection rate of L.major in P.
papatasi under the influence of bacteria (27). Notwithstanding these studies,
there are a few reports available on the micro flora of P. papatasi (24, 28).
A high prevalence of microbial infection in the digestive tract of wildcaught P. papatasi females was suggested to have a negative effect on
Leishmania transmission in endemic areas (29).
There is a new vector borne disease control method called paratransgenesis
that leads to reduce pathogen transmission by an insect vector (30). So,
microbes particularly bacteria presenting in insects may have an important
role in the epidemiology of human infectious disease (31).
Sand flies bacterial flora has been investigated on the isolated or via culture
of bacterial gut content and were identified by the use of classical
bacteriology, cloning (29-30).
Little is known about the prevalence and diversity of sand fly microflora
colonizing. This information is important for development of vector control
strategies (32). It is now widely recognized that symbiotic microorganisms
of arthropods play a crucial role in the ecology and evolution of their hosts.
The aim of the present study was to isolate, identify and examine the
prevalence of bacteria in P. papatasi and P. sergenti in Hamadan through a
culture dependent methodology. Results of this study may lead to identify
appropriate candidate/s for paratransgenesis approach. Further experiments
are required to clearly delineate the vectorial role (passive or active) of sand
flies. This information is important for the development of new strategies
for possible vector control.
Materials and Methods
This is a descriptive cross-sectional study. In The city of Hamadan (33°59’35°48’ N and 47°34’-49°36’ E) the average minimum and maximum
monthly temperatures in January and June are -2.7 °C and 25.6 °C,
respectively. The average relative humidity ranges are from 25% in July to
71% in January.
Sand flies were collected weekly, using sticky traps (castor oil coated white
paper 20×32 cm) from the beginning (May) till the end of the active season
(October) (33). Traps were installed at 18:00 pm and run until 07:00 am the
following morning. Then traps containing insects were collected and
transported to the laboratory in Hamadan. Isolation of sand fly guts was
conducted in a sterile environment under a microbiological lab hood on a
sterile glass slide. Before dissection, individual flies were surface sterilized
for 2 min in 70% ethanol. The gut from each sand fly was micro-dissected
and homogenized in test tubes (34). For species identification, sand flies
were mounted in Puriˊs medium and identified after 24 h, using the keys of
Mesghali et al., Nadim and Smart (35-37). The specimens were identified
based on morphological characters of the head and the abdominal
terminalia. Using rest of sand flies body for Bacterial Identification under
sterile conditions.
Bacterial Identification
The samples were serially crushed using a glass pestle in microtube to
incubate bacteria in Plate Count Agar, Mac Conkey agar and EMB. Plates
were then incubated at 37 ͦC for 24-48 h.
The initial identification of bacterial species was based on the colony
characteristics (involving colony size, shape, color, margin, opacity,
elevation and consistency) and the morphology of isolates based on Gram’s
staining procedure.
Pure cultures for each microbe were used for further identification
procedure. All of the isolates were differentiated by standard gram staining
and morphotypes. Mac Conkeys Agar,is a special selective medium for
gram negative bacteria (28, 38-39).
Finally, the API identification kit (API 20E, BioMerieux) was used for final
identification of Gram-negative bacteria. The identification of Grampositive bacteria was performed using the API Staph, API 20 Strep and
API50CH B following the manufacturer’s recommendations.
The colonies with different phenotype were sub cultured sequentially to
obtain single colony of the microbes. The best growing colonies and the
most characteristic ones were picked up by sterile loop and subjected to
purification in the same isolation medium. Agar streak method was used for
purification process. A well separated colony from each isolate was picked
up on nutrient agar slopes and incubated at 35°C for 24 h. Purity was checked
by microscopic examination of the isolate using Gram stain. All cultures
were maintained under aerobic conditions.
Gram-positive isolates that were not able to grow on the MacConkeys Agar
medium, tested with manual laboratory examinations such as oxidase,
catalase, coagulase, novobiocin susceptibility tests and mannitol medium.
Bacteria were isolated after 24-48 h. Gram stain: Jensen's modified method
was applied using crystal violet as a basic dye and safranine as counter stain
(40). The sterilization efficiency was controlled during the whole procedure.
Total colony counts were recorded for each sample and the average for
every sample was calculated.
Statistical analysis: All analyses were performed by SPSS-16 software.
Among the all processed sand flies, only 4 of them (3 males and 1 female)
were negative for bacteria. Eight bacterial strains were isolated from the
processed Phlebotominae sand flies.
The bacterial isolates corresponded to eight bacterial taxa: Acinetobacter
lwoffii, Pseudomonas aeruginosa, Enterobacter cloacae, Edvardsiela sp.
and Proteus mirabilis )Gram-negative bacteria) as well as Bacillus subtilis,
Staphylococcus saprophyticus and Micrococcus luteus (Gram-positive
bacteria) (Figure 1.).
Fig1. The percentage of each bacteria in sand flies
The presence of bacteria in insects can strongly influence their hosts’
biology. Information on the biological interactions between bacteria of sand
flies and Leishmania parasites they transmit is somewhat limited. However,
the differences in susceptibility to leishmanial infection based on
geographical distribution of sandflies were studied by many authors (41-42).
It is important to consider the microorganisms in vector insects, The present
study revealed that P. papatasi and P. sergenti female and male harbored
both Gram–negative and Gram–positive bacteria in their body.
In this survey, 200 insects were screened, and 8 bacterial species were
isolated and identified. These species were: Acinetobacter lwoffii,
Pseudomonas aeruginosa, Enterobacter cloacae, Edvardsiela sp. and
Proteus mirabilis )Gram-negative bacteria) as well as Bacillus subtilis,
Staphylococcus saprophyticus and Micrococcus luteus (Gram-positive
bacteria). Hassan and et al. isolated 4 species of Gram–negative bacteria
Staphylococcus haemolyticus and Neisseria mucosa of P. papatasi collected
from North Sinai and P. langeroni collected from El Agamy (43).
The most frequently isolated bacteria species of P. papatasi and P. sergenti
in the present study were Acinetobacter lwofii and Pseudomonas
aeruginosan. But in one study in Egypt B. thuringiensis was the most
frequently isolated bacteria species of P. papatasi (38). Dillon et al.
reported that the predominant bacteria species in the P. papatasi caught of
Sinai, Egypt were Enterobacter cloacae, E. sakazaki and Aeromonas sobria
(order Enterobacteriaceae) (25).
Although we used a nonselective medium to promote growth a wide range
of bacteria, due to not using specific media and culture conditions (e.g. an
aerobic condition) the medium generally favored in growth of gram negative
bacteria. Similarly, almost all of the studies analyzing the sand fly for
bacterial communities have also relied on culture dependent techniques in
their analyses where gram negative bacteria constituted the majority of their
findings (28, 44).
In addition, several studies have reported a higher prevalence of Gram
negative bacteria than Gram-positive ones in different vector insects (31,
45). This is in agreement with our observation that the majority of the
bacterial strains isolated in the present study were Gram-negative bacteria.
In addition, a correlation between the type of microbial flora detected and
the area inhabited by the sand fly has been showed by Hillesland and et al.,
where flies collected from the same region harbored almost the same kinds
of bacteria (44). Mukhopadhyay and et al. carried out a survey to study the
abundance of different natural flora of P. papatasi in different habitats of
Tunisia, Turkey, India and Egypt. They found variation in the species and
abundance of flora in sand flies collected from different habitats (28). These
results fit with our study. Therefore, it was suggested that flora diversity
more or less is a reflection of the environment where the sand fly resides.
Bacillus subtilis is a Gram-positive bactria which found in the present
study. Mukhopadhyay and et al. succeeded in introducing of B. subtilis as
candidate species for paratransgenesis due to the ability of this bacterium in
induction of sand fly oviposition behavior and real function of this as
symbiont and not merely as environmental contaminant (28).
Also Proteus species is found in many animals including insects (46-47).
Interestingly, in maggots, the bacteria P. mirabilis secrete antibacterial
toxins that kill other microbes but do not harm the maggots. Proteus
mirabilis is also highly resistant to the action of antimicrobial peptides, such
as polymyxin. It referred to the active anti-bacteria constituents as
“mirabilicide”(46). The presence of P. mirabilis in other studies showed that
this microorganism was considered beneficial (46-47). However, these data
have to be confirmed in the future by further studies carried out on more
The lack of Wolbachia isolate in the present study might be due to the
isolation and characterization methodology that we have used.
Further studies examining bacteria species in sand flies are needed to reveal
the relationship between bacteria and phlebotominae hosts.
Whether or not, the resident microbiota as a micro ecological factor can
regulate the prevalence of sand flies with transmissible infections need to be
investigated. Also, more investigation need to find the most effective
bacteria which can be used as bio-agent for combating Leishmania parasites.
In nature, despite the probable well balanced associations between some
bacteria and sand flies, there could be natural selective pressure involving
some species of bacteria, Leishmania and their vectors.
One study showed that the importance of considering the host microbiota as
an “extended immune phenotype” in addition to the host immune system
itself provide a unique perspective to understanding insects in health and
disease (48). Other study Showed that the capacity of bacteria to decrease
viral and parasitic infections in mosquito and tsetse fly vectors by activating
their immune responses or directly inhibiting pathogen development (49).
This could be happen in the sand fly and may lead to a reduction in
Leishmania infection within the sand fly host. Alder and Theodor (50) were
the first to suggest that the presence of other microorganisms might prevent
the development of Leishmania spp. in the sand fly. Dillon and et al.
showed that the Leishmania parasites often grow poorly in competition with
bacteria in P. papatasi , probably because of their relatively slow generation
time (25).
There is urgent search for new strategies to control major human parasitic
diseases, it might include engineering transgenic insects to reduce parasite
transmission. It is hoped that these bacteria may be able to be used as a
system to decrease vector-borne-diseases and to reduce the transmission of
diseases in the future.
Bacteria are increasingly seen as an important factor for modulating vector
competence in insect vectors so the presence of the bacteria in P. papatasi
and P. sergenti were discussed. However a depth research on the
interactions between sand fly and their bacteria and Leishmania is required.
We thank Dr. Amir Hossein Maghsood and Dr. Masoud Saidijam members
of Medical Sciences/University of Hamadan for their valuable assistance.
Motahare Mirhosseini, Dariush Bahrami and Saeed Porkeyhan to help for
research performing. This project received financial support from vicechancellor of research Deputy of Hamadan University of Medical Sciences
under the code 9211013716.
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Fig1. The precentage of each bacteria in sand flies

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