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Am. J. Trop. Med. Hyg., 79(6), 2008, pp. 881-886
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

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Identification of Aerobic Gut Bacteria from the Kala Azar Vector, Phlebotomus argentipes: A Platform for Potential Paratransgenic Manipulation of Sand Flies

Heidi Hillesland{dagger}, Amber Read{dagger}, Bobban Subhadra, Ivy Hurwitz, Robin McKelvey, Kashinath Ghosh, Pradeep Das, AND Ravi Durvasula*
Department of Internal Medicine, University of New Mexico, Albuquerque, New Mexico; Walter Reed Army Institute of Research, Silver Spring, Maryland; Rajendra Memorial Research Institute of Medical Sciences, Patna, India


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Visceral leishmaniasis is an understudied parasitic disease responsible for significant global morbidity and mortality. We are presently investigating a method of disease prevention termed paratransgenesis. In this approach, symbiotic or commensal bacteria are transformed to produce anti-Leishmania molecules. The transformed bacteria are delivered back to sand flies to inactivate the parasite within the vector itself. In this study, we identified 28 distinct gut microorganisms from Phlebotomus argentipes trapped from four visceral leishmaniasis–endemic sites in India. A significant percent of Staphylococcus spp., environmental bacteria, and Enterobacteriaceae were identified. Two non-pathogenic organisms, Bacillus megaterium and Brevibacterium linens, were also isolated. Both organisms are also used extensively in industry. Our results indicate that B. megaterium and B. linens are possible candidates for use in a model of paratransgenesis to prevent transmission of Leishmania.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leishmaniasis is a global health concern with an estimated 12 million people infected and 367 million at risk. This disease is caused by the protozoan kinetoplastid flagellate Leishmania donovani in India, L. infantum in Europe, and L. chagasi in South America. Natural transmission occurs through the bite of an infected sand fly of the genus Phlebotomus (Old World) or Lutzomyia (New World). Visceral leishmaniasis, also known as kala azar, is perhaps one of the most devastating forms of leishmaniasis. Two hundred fifty thousand cases are reported in India annually and, of those, 90% are from the state of Bihar. The leading vector for kala azar is the sand fly P. argentipes.

Despite the demonstrated public health importance, relatively little work has been directed at disrupting the transmission of Leishmania parasites by the insect vector. DDT spraying associated with the National Malaria Control Program in India in the 1960s was very successful in reducing sand fly populations, and cases of kala azar were nearly eliminated.1 However, recent studies show that certain populations are gaining insecticide resistance,2–4 resulting in growing concern about the efficacy, cost, and environmental toxicity of insecticide use. In many parts of India, return of peri-domestic populations of sand flies has been observed within 1 year of insecticide spraying.5 Recent surveys in Patna, India, further showed rates of DDT resistance among P. argentipes ranging from 60% to 100% (Das P and others, unpublished data). Difficulties in sustaining insecticide campaigns over a number of decades, the attendant risks of toxicity, the evolution of vector resistance, and the lack of effective vaccines to protect against kala azar necessitate the development of alternative methods to block transmission of L. donovani.

Our group has been developing a novel paratransgenic strategy to control vectorial transmission of certain infectious agents. In this strategy, commensal or symbiotic bacteria found at mucosal sites of transmission are isolated and genetically altered to elaborate molecules that kill the infectious agents. Transgenic bacteria are delivered back to insect vectors to block pathogen transmission. Our laboratory initially developed this "Trojan Horse" approach to combat transmission of the Chagas disease parasite, Trypanosoma cruzi, by a triatomine vector. In the Chagas disease vector, Rhodnius prolixus, the symbiotic bacterium, Rhodococcus rhodnii, was transformed to produce the anti-trypanosomal peptide cecropin A at levels capable of eliminating T. cruzi carriage by the bug.6 Subsequently, a paratransgenic system was developed to express a functional antibody in the triatomine bug.7 We have since applied this approach to commensal bacteria of the human respiratory tree8 and sharpshooter vectors of Pierce’s disease.9

For the paratransgenic approach to be successful in the sand fly, the identification of suitable extracellular commensal microorganisms within the vector is critical. Although relatively sparse, some literature exists on the gut microbiology of field-caught sand flies. Dillon and others10 evaluated gut microbes from P. papatasi caught in Egypt. Microbes isolated from these studies included E. coli, species of Pseudomonas, Enterobacter, Acinetobacter, Erwinia, and Propionibacterium in addition to unspeciated gram-negative cocci, gram-positive cocci, and gram-positive rods. Rajendran and Modi11 evaluated microorganisms from field-caught P. argentipes and Sergentomyia spp. from cattle sheds in India and from laboratory-reared P. papatasi. They reported isolation of E. coli, Bacillus spp., Staphylococcal spp., Micrococcus tetragenes, and yeast. Although these studies established presence of bacteria in the digestive tract of sand flies, they consistently yielded a variety of pathogenic bacterial species.

The paratransgenic approach would deploy commensal sand fly bacteria that are non-pathogenic to humans or animals. Such non-pathogenic bacteria would be transformed to deliver peptides with anti-leishmanial activity. One potential strategy for delivery of the transformed microbes is at sand fly breeding sites. These sites in India have been characterized previously and are found in association with cattle sheds that are often in close proximity to human dwellings.12

Here we report a microbiologic analysis of the gut flora of P. argentipes from four kala azar–endemic regions of Bihar state in India. We have identified several non-pathogenic commensal bacteria that could have potential use for paratransgenic manipulation of P. argentipes. Two of these, Bacillus megaterium and Brevibacterium linens, are extensively used in commercial applications in Bihar and could serve as delivery vehicles to block vectorial transmission of L. donovani.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sand fly collection. Sand flies were collected from four different regions within the state of Bihar, India. The geographic locations of the sampling areas were Gaya (24.79 N, 85.01 E), Gulmehiya (25.37 N, 85.13 E), Bahapur (24.53 N, 85.07 E), and Mahnar (25.66 N, 85.44 E). CDC light traps were placed in cowsheds between the hours of 4:00 AM and 9:00 AM during the month of October. In each case, samples were located immediately adjacent to human dwellings. Sand flies were narcotized, removed from the traps, and sorted by species. Only P. argentipes were examined in this work. Insects from each locality were pooled in separate tubes and stored at –70°C until further use.

Isolation of commensal microbes. Aerobic gut bacteria were isolated from each sand fly by one of the following two methods. In initial experiments, the gut from each sand fly was microdissected from the insect and homogenized in 100 µL of 0.1% peptone. A portion of this homogenate was plated onto brain heart infusion (BHI) plates. In subsequent experiments, each sand fly was surface sterilized for 30 seconds in ethanol, rinsed thoroughly in phosphated-buffer saline (PBS) and transferred to a 1.5-mL microcentrifuge tube. The insect was homogenized in 30 µL of BHI. An aliquot of this homogenate was plated onto BHI plates. BHI was chosen as a nonselective medium to promote growth of a diversity of microbes including nutritionally fastidious bacteria. Plates from both isolation methods were incubated at 28°C for up to 2 weeks. To assess microbial growth the total number of total colony-forming units (CFUs) was determined. The number of phenotypically different colonies was counted to determine the diversity of microbes in each sand fly. In a subset of samples, the number of phenotypically similar colonies was counted to assess the relative abundance of each microbial species per sand fly. Pure cultures for each microbe were obtained from these primary plates. All of the isolates were differentiated by gram staining and morphological shape.

Identification of microbes. Genomic DNA was isolated from each of the pure cultures. 16S rDNA PCR amplifications were performed from these samples using the forward primer 5'-AGAGTTTGATGGCTCAG-3' and the reverse primer 5'-TACGGCTACCTTGTTACGACTT-3'. Thermal cycling reactions consisted of an initial denaturation (95°C, 2 minutes) followed by 35 cycles of denaturation (95°C, 15 seconds), annealing (65°C, 30 seconds), and extension (72°C, 60 seconds), with a single final extension (72°C, 2 minutes). To control for the presence of contaminating nucleic acids, water samples with no template DNA were run in parallel. The amplicons were purified from gels and sequenced using the BigDye Terminator V2.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) using the PCR primers as described above as well as an internal sequence primer, 5'-GTGCCAGCCGCCGCGG-3'. Ambiguities were re-sequenced, and at least 98% percent of the complete double-stranded sequences were obtained. Sequences were aligned using the Sequencer 4.8 software (Gene Codes). Completely aligned sequences were compared with the BLAST database. Isolates were identified when their 16S rDNA sequences shared ≥ 97% homology with completed 16S rDNA sequences found in the GenBank database.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In this analysis, 109 P. argentipes were examined (Table 1Go). Although 103 of these insects were collected from four kala azar–endemic regions in the state of Bihar, India, the other six sand flies were collected from laboratory colonies.


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TABLE 1
Number of sand flies collected from each site
 
The total number of CFUs per sand fly ranged from 0 to < 20. As shown in Table 2Go, the majority of the sand flies examined harbored microorganisms (59%). A significant number, however, did not harbor any organisms (41%). Among sand flies that harbored bacteria, polymicrobial cultures (38%) were more common than monocultures (21%). In one case, six different microbes were isolated from a single sand fly.


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TABLE 2
Number and percentage of microbes isolated from each sand fly
 
In cases of polymicrobial colonization, the relative abundance of each microbe was examined in 48 of the sand flies. This analysis is shown in Table 3Go. Our results show that when present with other organisms, Staphylococcus spp., Escherichia coli, and Enterobacter spp. were generally found in greatest abundance. Bacillus spp. and Brevibacterium linens were usually found in least abundance.


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TABLE 3
Relative abundance of microbes isolated from a sample of sand flies when polymicrobial colonization was present
 
We isolated > 100 different microbial colonies from the sand flies. These isolates were subsequently identified by 16S rDNA sequencing. A total of 28 distinct microorganisms were identified from the gut of P. argentipes. Microorganisms from each collection site are tabulated in Table 4Go. Some overlap can clearly be seen between microbes isolated from the four different sites. Gram-negative Enterobacteriaceae such as E. cloacae and E. coli were isolated from sand flies collected from all four sampling sites, as were a number of soil organisms. Pathogenic staphylococci were isolated from three of four sites. Importantly, however, several non-pathogenic organisms, namely B. megaterium, B. linens, and related species, were also isolated from all four sites. It is also interesting to note that all the sand flies collected from the site in Bahapur harbored the same microbes, and seven of nine sand flies from the site in Mahnar carried Brevibacterium casei, suggesting a strong correlation between sand fly–microbial association and the environment in which they reside.


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TABLE 4
Bacteria isolated from sand flies by location in Bihar, India
 
Table 5Go classifies all the isolated bacteria into groups that are known to be pathogenic and non-pathogenic to humans. It is critical that a non-pathogenic organism be chosen for our proposed application of paratransgenesis.


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TABLE 5
Pathogenicity of bacteria isolated from the gut of P. argentipes
 
Table 6Go shows further regional differences and similarities. Enterobacteriaceae tended to be more prevalent in Gaya and Bahapur, whereas Bacillus megaterium was isolated most commonly from sand flies collected in Gulmehiya. Sand flies from Gaya had the most B. linens, whereas sand flies from Bahapur more commonly harbored other species of Brevibacterium. Table 6Go looks specifically at B. linens and B. megaterium because of their lack of pathogenicity and potential application in future paratransgenesis work.


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TABLE 6
Percentage of sand flies carrying specific microbes by region and total
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study is the most comprehensive evaluation of the microbiology of P. argentipes to date. Our results showed that P. argentipes isolated from kala azar–endemic regions in Bihar harbor a variety of gut bacteria. The diversity of microbes would strongly suggest that the sand fly–microbial association reflects the environment in which these sand flies reside. For example, a number of the isolates identified in our work and by others showed that Enterobacteriaceae are prevalent in P. argentipes. Kesari and others12 identified sand fly breeding sites by cowsheds. Adult sand flies are also known to rest on the walls in the dark corners of cowsheds. It is therefore highly likely that these sand flies acquire Enterobacteriaceae from cow feces. There were a large number of soil and environmental bacteria isolated in this study, such as species of Acinetobacter, Gordonia, Microbacterium, Micrococcus, Stenotrophomonas, and Bacillus. The isolates showed regional variation. This suggests that the local soil and water environment plays an important role in colonization of the sand flies with regional bacteria encountered at breeding sites or during sugar meals.

Previous studies of sand fly gut microbiology yielded similar profiles of bacteria. Dillon and others10 showed that Enterobacteriaceae such as species of Enterobacter were isolated in greatest abundance from P. papatasi. We also found a significant percentage of Enterobacteriaceae (13%), although Staphylococcus spp. were found in greatest abundance (21%). It has been reported previously that species of Enterobacter are the most common bacteria isolated from the gut of insects.13 E. coli, Bacillus spp., Micrococcus spp., and coagulase-negative Staphylococcus spp. were previously reported by Rajendran and Modi.11 These organisms were also isolated in our study.

It was additionally evident that a high percentage of sand flies did not harbor bacteria. This finding was similar to previous reports,10 suggesting that our sterilization and homogenization technique was adequate. Sand flies do not have known symbiotic bacterial associations. It is therefore not surprising that the sand flies are variably colonized with bacteria. In addition, it is likely that colonization rates under field conditions are greater than reported because bacteria were not cultivated in anaerobic conditions, and certain environmental bacteria will not grow on any media and may not be identified.

We identified a number of bacterial pathogens and potential pathogens in our study. We embarked on this microbiologic survey to identify bacteria that might be used in future work with paratransgenesis in an effort to block vectorial transmission of L. donovani. Although species such as Enterobacter cloacae have been used as shuttle systems to deliver, express, and spread foreign genes in termite colonies,14 it is important that pathogenic bacteria not be used in our work. As part of the paratransgenic approach, it will be necessary to deliver transformed bacteria to the sand flies to allow for sand fly colonization. There is a close association of humans and sand flies in domestic and peri-domestic areas, necessitating the use of non-pathogenic bacteria for future work. Importantly, the extensive nature of this study allowed for identification of a number of non-pathogenic bacteria that could be used as transformants in a paratransgenic approach.

Two bacteria isolated were of particular relevance: B. megaterium and B. linens. Although the association rates of these organisms with the sand flies are low, it should be noted that both organisms are extensively used in biotechnological operations for the production of various compounds and are known to be safe. B. megaterium is marketed as a crude bio-fertilizer and used extensively in many regions of Bihar. Recent studies have shown that B. megaterium has the ability to promote growth and reduce disease in different Indian cultivable plants.15 In addition, both B. linens and B. megaterium have been reported to have a probiotic effect on many animals.16,17 B. linens is one of the main bacteria used in industrial cheese ripening. Some of the sand flies from our collection were from areas with intimate household dairy operations like milk curdling, cheese, and ghee preparation. Methanethiol and isovaleric acid, produced by B. linens, are very strong insect attractants and have been shown to raise the number of mosquitoes and other insects in olfactometer traps by two to three times.18,19 This may be useful in encouraging colonization of sand flies with transformed B. linens in a paratransgenic application.

Transformed B. megaterium and B. linens would be deployed in large concentrations in sand fly breeding sites to selectively colonize the vectors. By increasing the concentration of these bacteria in the soil, we hope to increase the degree of colonization of sand flies despite the low natural rates of colonization seen in our study. Many of the sand flies in the study were colonized with multiple bacteria. Our ongoing studies involve transforming at least two species of bacteria to potentially improve colonization rates of transformants in the sand flies.

Because B. megaterium and B. linens currently play an integral role in agriculture in Bihar, it is conceivable that transformed B. megaterium and B. linens can additionally be spread through a combination of various operations related to agriculture. The marketing of transformed B. megaterium as a bio-fertilizer formula through the proper regulation from Indian health and agricultural departments may be a viable method of spreading transformed bacteria in this endemic region. Furthermore, because most of the sand fly breeding grounds are in close proximity to cattle and dairy operations, another approach could involve release of transformed B. linens as a probiotic for cattle feed. We hope to overcome low percentage rates of colonization and variability of bacterial colonization by using several delivery methods to effectively colonize sand flies with transformed bacteria.

The presence of B. linens and B. megaterium therefore opens a new avenue for vector control of kala azar. Transformation of these species of bacteria to express L. donovani–blocking molecules and the transient expression of these molecules at the critical parasite developmental period in the sand fly vector could potentially serve as an effective platform for paratransgenic control of L. donovani transmission. A large number of anti-leishmania molecules are available, such as histatin 5, racemoside A, monoclonal antibodies, and temporins.20–26 For paratransgenesis, the molecule must be selectively toxic to the amastigotes and promastigotes and pose no environmental threat. Additionally, the molecule must not inhibit growth of the bacteria that express it.

We have recently transformed B. megaterium to express a marker single-chain antibody. Studies are currently underway to track the persistence of this genetically modified microbe through the developmental stages of P. argentipes (unpublished data; data not shown). To date, little is known about transtadial passage of bacteria in sand flies Volf and others27 suggested that there is colonization that persists through the larval stages to the emergent adult sand fly. This would allow for delivery of transformed bacteria in large numbers to the breeding sites to encourage selective colonization of the various developmental stages of the sand fly with resultant colonization of the adult emergent sand fly before ingestion of a blood meal.

This study provided the most extensive survey of sand fly gut microbes to date. Colonization trends were similar to those previously reported in sand flies. This study additionally allowed for broader identification of microbes including non-pathogenic species. Two bacterial species identified, B. megaterium and B. linens, are ideal targets that could be used toward paratransgenic control of Indian leishmaniasis. Leishmaniasis causes significant mortality and is an understudied disease. Our work is ongoing, and we are hopeful that the data presented here will serve as an effective platform for future efforts to prevent leishmaniasis.

Received May 29, 2008. Accepted for publication August 27, 2008.

* Address correspondence to Ravi Durvasula, Department of Internal Medicine, University of New Mexico, Albuquerque, NM 87131. E-mail: ravi.durvasula{at}va.gov Back

{dagger} These authors contributed equally to this manuscript. Back

Authors’ addresses: Heidi Hillesland, Amber Read, Bobban Subhadra, Ivy Hurwitz, Robin McKelvey, and Ravi Durvasula, Department of Internal Medicine, University of New Mexico, Albuquerque, NM 87131. Kashinath Ghosh, Walter Reed Army Institute of Research, Silver Spring, MD 20910. Pradeep Das, Rajendra Memorial Research Institute of Medical Sciences, Patna, India 800007.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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