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Phylogenetic characterization of bacteria in the gut of house flies (Musca domestica L.)

Arvind K. Gupta, Dana Nayduch, Pankaj Verma, Bhavin Shah, Hemant V. Ghate, Milind S. Patole, Yogesh S. Shouche
DOI: http://dx.doi.org/10.1111/j.1574-6941.2011.01248.x 581-593 First published online: 1 March 2012


House flies (Musca domestica L.) are cosmopolitan, ubiquitous, synanthropic insects that serve as mechanical or biological vectors for various microorganisms. To fully assess the role of house flies in the epidemiology of human diseases, it is essential to understand the diversity of microbiota harbored by natural fly populations. This study aimed to identify the diversity of house fly gut bacteria by both culture-dependent and culture-independent approaches. A total of 102 bacterial strains were isolated from the gut of 65 house flies collected from various public places including a garden, public park, garbage/dump area, public toilet, hospital, restaurant/canteen, mutton shop/market, and house/human habitation. Molecular phylogenetic analyses placed these isolates into 22 different genera. The majority of bacteria identified were known potential pathogens of the genera Klebsiella, Aeromonas, Shigella, Morganella, Providencia, and Staphylococcus. Culture-independent methods involved the construction of a 16S rRNA gene clone library, and sequence analyses supported culture recovery results. However, additional bacterial taxa not determined via culture recovery were revealed using this methodology and included members of the classes Alphaproteobacteria, Deltaproteobacteria, and the phylum Bacteroidetes. Here, we show that the house fly gut is an environmental reservoir for a vast number of bacterial species, which may have impacts on vector potential and pathogen transmission.

  • 16S rRNA gene
  • culture dependent
  • clone library
  • culture independent
  • bacterial diversity


Dipteran flies are one of the most abundant and important groups of insects, serving as vectors for some of the most devastating diseases affecting humans. The higher Diptera includes the family Muscidae, whose most well-known members are commonly called house flies (Musca domestica L.; Skidmore, 1985). House flies reproduce and develop in decaying organic matter such as animal manure, human refuse, open privies, soiled animal bedding, litter, and waste around food and vegetable processing plants, which all are areas teeming with diverse and active microbial communities (Greenberg, 1973; Graczyk et al., 2001; Moon, 2002). All trophic levels of house flies (e.g. larvae, pupae, adults) are commonly contaminated with various microorganisms. As adult house flies are highly mobile, they transport bacteria from septic environments to other substrates via contamination of their surfaces (feet, wings, bodies) and also by regurgitation of crop contents (Moon, 2002). Their persistent and ubiquitous association with humans, animals, food, refuse, and excreta makes flies potential mechanical or biological vectors for the dissemination of pathogenic and multidrug-resistant bacteria (Zurek et al., 2000; Graczyk et al., 2001; Alam & Zurek, 2004; Rahuma et al., 2005; Macovei & Zurek, 2006; Macovei et al., 2008; Chakrabarti et al., 2010; Ahmad et al., 2011).

House flies have been implicated in the transmission of serious diseases such as anthrax, ophthalmia, typhoid fever, tuberculosis, cholera, and infantile diarrhea (Scott & Lettig, 1962; Greenberg, 1965; Keiding, 1986) and have been demonstrated to harbor or transmit other pathogenic bacteria including Salmonella spp. (Greenberg, 1971), Proteus spp., Shigella spp. (Greenberg, 1971), Chlamydia spp., Campylobacter jejuni (Shane et al., 1985), Klebsiella sp. (Fotedar et al., 1992), Escherichia coli O157:H7 (Kobayashi et al., 1999; Ahmad et al., 2007), Yersinia pseudotuberculosis (Zurek et al., 2001), and Helicobacter pylori, the causative agent of gastric ulcer (Li & Stutzenberger, 2000). Recently, these insects were reported to be involved in disease outbreaks including E. coli O157:H7 (Sasaki et al., 2000) in Japan and Vibrio cholerae in India (Fotedar, 2001).

The association of living bacteria within the alimentary canal or/and on the body surface of house flies, and their transmission has been demonstrated through various studies. Historically, the first detailed observations of this nature were made by Graham-Smith (1910) who caught flies randomly, artificially infected them with pathogenic bacteria, and recorded their recovery over time. Early studies by Mcguire & Durant (1957) found approximately 20 times more internal than external bacteria in M. domestica, denoting on the role of spatial location of microorganisms in their survival. The study of microbial ecology of the insect gastrointestinal tract is experiencing a revival owing to the development of molecular techniques for studying complex microbial communities. Prior to our study, knowledge of the house fly-associated microbiota was limited to culture-dependent assays (employing culture alone or culture followed by PCR) and focused only limited or even single species (Nayduch et al., 2001; Alam & Zurek, 2004; Szalanski et al., 2004). However, it has been suggested that < 1% of bacterial species can be cultivated (Staley & Konopka, 1985), and thus culture-dependent studies of house fly microbial ecology may be insufficient at best.

Interestingly, despite its great medical importance, there have been few studies that fully assess the nature and diversity of the microbial community associated with house flies. There have been several qualitative reports of microbial community isolated from house fly surveys (Sulaiman et al., 2000; Nazni et al., 2005; Rahuma et al., 2005; Vazirianzadeh et al., 2008; Butler et al., 2010). However, there is a lack of studies characterizing bacteria housed in alimentary tract of the house fly. Therefore, in this study, we planned to characterize the total gut associated microbiota of house flies collected from various public places that represented both sanitary and unsanitary areas. These sites included a garden, public park, garbage dump, public toilet, hospital, restaurant/canteen, mutton shop/market, and house/human habitation. We utilized both culture-dependent (i.e. live culture followed by 16S rRNA gene sequencing) and culture-independent (i.e. total DNA extraction, PCR, sequencing of cloned 16S amplicons) approaches (Pidiyar et al., 2004). To the best of our knowledge, this is the first detailed study, which utilizes 16S rRNA gene sequence analysis for surveying house fly gut bacteria. The 16S rRNA gene sequences generated by both methods revealed a wide variety of new and unreported potential pathogenic bacteria associated with the gut of adult house flies. Results of this study can shed light on the possible role of flies as both vectors and environmental reservoirs for human pathogenic bacteria.

Materials and methods

Sample collection

A total of 65 adult house flies were independently captured using aerial insect nets from public places including (n for each site appears in parentheses): a garden (8), public park (8), garbage dump (8), public toilet (8), hospital (7), restaurant/canteen (8), mutton shop/market (8), and human residential habitation (10). All locations were proximal to the National Centre for Cell Science in Pune, Maharashtra, India (Latitude: 18° 34′ N, Longitude: 73° 58′ E). Sites were selected owing to the observed abundance of adult house flies and existence of ecological conditions enhancing their survival and persistence. House flies were collected during the rainy season (August–September 2008), with the average days being warm and sunny with temperature ranging from 25 to 30 °C, allowing ample fly activity. Flies were immediately transferred from the aerial insect net to zip-locked plastic bags and brought to the laboratory within 45 min, where they were either processed immediately or stored separately in 15-mL falcon tubes containing 2 mL sterile 0.85% NaCl (w/v) at 4 °C till further processing. Flies were anaesthetized using ethyl acetate and then surface-sanitized with 70% ethanol for 5 min followed by washing in sterile phosphate-buffered saline (PBS) before dissections. Legs, wings, abdomens, and guts were microscopically dissected under sterile conditions and transferred individually to 1.5-mL microfuge tubes containing 500 μL of sterile PBS. Genomic DNA was extracted from legs, wings, and abdomens to confirm M. domestica species identity after sequencing PCR products of mitochondrial 16S rRNA (16S2) and cytochrome oxidase I (COI) genes. Fly guts were sonicated for 30 s, macerated with a plastic pestle, and then vortexed at medium speed for 2 min to separate bacterial cells from the gut wall. The gut homogenates obtained from flies from the same site were pooled and were used: (1) to inoculate growth media for bacterial colony analysis (culture-dependent identification) and (2) for concurrent total DNA extraction and PCR to amplify the 16S rRNA genes of microbial community (culture-independent identification) and characterized them based on their sequences.

Bacterial cultures from house fly gut homogenate

A 100-μL aliquot of the gut contents was serially diluted up to 10−6 and plated on Luria–Bertani (LB) agar, Tryptic Soy agar (TSA), and blood agar base with 10% (v/v) human blood (HiMedia, India). Cultures were incubated aerobically at 28 °C for 18–72 h. Bacterial colonies that were morphologically distinct were selected from each culture plate for further characterization. These colonies were restreaked on LB agar plates until pure culture was obtained, and isolates were preserved in 50% (v/v) glycerol/LB broth at −80 °C for subsequent DNA extraction.

DNA extraction from house flies, bacterial cultures, and gut homogenate

For species identification confirmation, genomic DNA was extracted from legs, wings, and abdomens of each collected fly using DNeasy Blood and Tissue Kit (Qiagen) as described in the manufacturer's protocol. Pure bacterial isolates from the house fly gut homogenate were subcultured in 5-mL LB broth at 28 °C for 48 h. Cell suspensions were lysed using CTAB and proteinase K at 37 °C for 1 h. Chromosomal DNA was isolated by the standard phenol/chloroform/isoamyl alcohol (25 : 24 : 1) extraction and isopropanol precipitation method (Sambrook et al., 1989) for PCR amplification. For culture-independent identification of gut microbiota, total bacterial community DNA was extracted from remaining gut homogenate (~ 400 μL) using a DNeasy Blood & Tissue Kit (Qiagen) with the following modifications to the manufacturer's protocol: (1) lysozyme (100 μL per sample, 10 mg mL−1) was added to the gut homogenate and incubated for 2 h at 37 °C and (2) samples were frozen at −80 °C for 5 min and heated to 65 °C for 10 min (repeated three times), followed by centrifugation at 12 000 g for 5 min, and addition of proteinase K (20 μL per sample, 20 mg mL−1) with subsequent incubation at 37 °C for 30 min. Integrity of the extracted DNA was assessed by 0.7% agarose horizontal gel electrophoresis in TAE buffer (40 mM Tris, 20 mM acetate, 2 mM EDTA) and visualized by ethidium bromide staining. The concentration of extracted DNA was checked on NanoDrop ND-1000 spectrophotometer (NanoDrop Biotechnologies) and ranged between 200 and 300 ng μL−1.

PCR amplification of DNA extracted from cultured isolates and gut homogenate

The 16S rRNA gene was amplified from DNA extracted from cultured isolates using universal bacteria-specific primers: 16F27 (5′-CCAGAGTTTGATCMTGGCTCAG-3′) and 16R1525XP (5′-TTCTGCAGTCTAGAAGGAGGTGWTCCAGCC-3′) (Pidiyar et al., 2004). Alternatively, primers 530F (5′-GTCCCAGCMGCCGCGG-3′) and 1490R (5′-GGTTACCTTGTTACGACTT-3′) (Weisburg et al., 1991) were used for the amplification of DNA extracted from the gut homogenate. PCR amplification was carried out using previously reported temperature settings and cycling parameters (Weisburg et al., 1991) in a 25-μL reaction mixture containing 200 μM (each) dNTPs, 1 μL of a 10 μM each primer, 1 μL of (~ 50 ng) DNA template, and 2.5 U of Taq DNA polymerase (Bangalore Genei, India) with 1× reaction buffer supplied by the manufacturer. Two microliters of amplified DNA was examined using 1% agarose horizontal gel electrophoresis in TAE buffer. Amplified PCR products from gut homogenates of house flies from different collection sites were pooled and subsequently purified with a PCR purification kit (Qiagen), following the manufacturer's instructions.

16S rRNA gene clone library construction, screening, and sequencing

The 16S rRNA gene amplicon library was constructed from the gut homogenate by ligating purified PCR products into pGEM-T easy vector (Promega) according to manufacturer's instructions. Ligation mixture was transformed in chemically competent E. coli JM109 cells with 30-min recovery time. Transformants were grown overnight on LB plates containing 100 μg mL−1 each of ampicillin, X-gal, and Isopropylβ-d-1-thiogalactopyranoside. Two hundred and fifty white colonies were randomly selected and screened for insert by PCR using vector-specific M13F (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) and M13R (5′-TCACACAGGAAACAGCTATGAC-3′) primers. PCR was performed by incubating the mixture at 94 °C for 7 min (to lyse the cells and initial denaturation) followed by 35 cycles each of 1 min at 94, 55 and 72 °C and a final extension step for 10 min at 72 °C. Clones with proper insert sizes were purified and sequenced in both directions using M13F and M13R primers. The primers used to obtain the sequence of 16S rRNA gene of the cultured isolates were the same as for PCR amplification (16F27 and 16R1525XP). An internal primer 16F536 (5′-GTGCCAGCAGCCGCGGTRATA-3′) was also used in addition to above primers, and sequences were determined with an ABI-PRISM 3730 DNA analyzer (Applied BioSystems Inc., Japan).

Sequence-based taxonomic and phylogenetic analyses

The 16S rRNA gene sequences were assembled and edited with ChromasPro version 1.5 software (www.technelysium.com.au/ChromasPro.html). Flanking vector sequences were trimmed from both the ends. Chimeric sequences and other anomalies were checked by bellerophon server (Huber et al., 2004), chimera_check v. 2.7 (Cole et al., 2003), and mallard software (Ashelford et al., 2006) using pairwise comparisons within a multiple alignment. Putative chimeras identified by the programs were cross-checked with BLASTn (Altschul et al., 1997) and compared with closest cultured sequences retrieved from the database. Suspected chimeras were excluded from further analysis. Multiple sequence alignments were performed using ClustalW version 1.8 (Thompson et al., 1994), and aligned sequences were edited and corrected manually using dambe (Xia & Xie, 2001) to generate an unambiguous sequence alignment. Two hundred and thirteen 16S rRNA gene sequences were selected on the basis of initial results and subjected to further phylogenetic analysis using neighbor-joining method implemented through DNADIST from the phylip version 3.61 (Felsenstein, 1993). Operational taxonomic units (OTUs) were generated using furthest neighbor algorithm of dotur program (Schloss & Handelsman, 2005). OTUs generated at 0.03 E.D. (Evolutionary Distance) or the OTUs formed by the sequences that present a similarity equal or > 97% (Stackebrandt & Goebel, 1994) were used for further analysis. One representative cultured isolate or a clone sequence from each OTU was taken for taxonomic assessment which was analysed using blast (Altschul et al., 1997) and megaBLAST (Zhang et al., 2000) programs against the EzTaxon database of type strains with validly published prokaryotic names (Chun et al., 2007). Phylogenetic dendrograms were constructed by neighbor-joining method using mega version 4.0 software (Tamura et al., 2007) to determine the relationship of these OTUs with known sequences of database. One thousand bootstrap replicates were generated, and a consensus tree was derived.

Biodiversity analysis based on dotur

For biodiversity analysis, Good's coverage was calculated using the formula [1 − (n/N)] × 100, where n is the number of OTUs with single isolate or clone sequence, and N is the library size (Good, 1953). Rarefaction curve was plotted as the number of OTUs vs. the number of isolate/clone sequences assuming that one OTU is formed by the sequences that show a similarity equal or > 97% (Hurlbert, 1971). Biodiversity index was determined at evolutionary distance (E.D.) of 0.03 or a sequence similarity value of 97% for bacterial population using the Shannon–Weaver Index (H′ = −Σ n/N ln n/N), which was used as a measure of relative diversity including richness and evenness; the Simpson Index [D = (Σn(n − 1))/N(N − 1)] of diversity (Weisburg et al., 1991) and the richness [Chao1, abundance-based coverage estimators (ACE) and Jackknife] were estimated as an alternative to Shannon diversity. A bar chart was constructed to compare the percentage distribution of cultured isolates and clone library sequences in different taxa.

Nucleotide sequence accession number

The 16S rRNA gene sequences obtained in this study have been deposited in the GenBank database under accession numbers HQ407007-HQ407321.


Fly identification

Species confirmation of wild caught flies as M. domestica L. was made both by morphological characteristics and by sequencing mitochondrial 16S rRNA (16S2) and cytochrome oxidase I (COI) genes (Simon et al., 1994; Krafsur et al., 2005).

Taxonomic distribution of 16S rRNA gene sequences

To examine the total gut bacterial diversity, we used high-throughput 16S rRNA gene–based sequencing approach. We identified 28 and 31 OTUs from the cultured isolates and the clone library sequences, respectively, which were taxonomically distributed to the phyla (Alpha-, Beta-, Gamma-, and Delta-) Proteobacteria, Firmicutes, and Bacteroidetes. The percentage distribution of cultured isolates and clone library sequences in different taxa is presented in Fig. . The majority of isolate (89.21%) and clone (59.62%) sequences belonged to the class Gammaproteobacteria. Isolates belonging to the classes Alphaproteobacteria, Deltaproteobacteria, and Bacteroidetes could not be recovered by our culture-based methods. List of all bacteria detected and their medical significance along with percent identity, collection sites, and methodology are shown in Table . We identified seven novel phylotypes showing ≤ 97% sequence similarity (Table ) that could represent new genera or species. A total of 41 different genera covering 55 different phylotypes were recovered by applying both methods (culture-dependent and culture-independent). Comparative studies revealed 10 genera, and three species (Wohlfahrtiimonas chitiniclastica, Acinetobacter soli, and Proteus mirabilis) common in cultured isolates and the clone library (Table ). This indicates that the culture-independent studies bolstered the cultured results with many additional genera. Interestingly, 12 genera found in cultured isolates were not identified in the clone library, whereas 19 different genera that were recovered from clone library were not seen in cultured isolates (Table ). These results imply that both culture-dependent and culture-independent methods are needed to unveil the total microbial diversity of house fly gut.

Percentage distribution of cultured isolates and clone library sequences in different taxa based on 16S rRNA gene sequences.

View this table:

List of bacteria identified based on taxonomic assessment of 16S rRNA gene sequences from the EzTaxon database and their summary of medical significance

Homologue identified (abundance)Percent identityCollection siteMethodMedical significance
Achromobacter ruhlandii (1)97.72NANot known
Acinetobacter bereziniae (2)98.68NAAcinetobacter spp. have been implicated in nosocomial infections
Acinetobacter haemolyticus (2)99.62M, R+
Acinetobacter radioresistens (1)96.93C+
Acinetobacter soli (1)99.79H+, −
Acinetobacter soli (7)97.88NA+, −
Aeromonas hydrophila (38)99. 80NAGastroenteritis and wound infections
Aeromonas veronii (3)100P, T+Opportunistic gastrointestinal pathogen
Bacillus amyloliquefaciens (1)99.57H+Not known
Bacillus firmus (32)99.44NANot known
Chryseobacterium haifense (1)96.98NANot known
Clostridium sordellii (2)98.56NAIsolated from the immunocompromised patient's stool
Comamonas testosterone (4)99.79NARarely implicated as a human pathogen
Cronobacter sakazakii (1)90.31C+Recognized as causative agent of neonatal bacteraemia, meningitis, and necrotizing enterocolitis
Desulfovibrio senezii (18)99.69NANot known
Dysgonomonas mossii (1)99.17NAIsolated from intestinal juice of a patient with pancreatic cancer
Enterobacter aerogenes (3)97.11G, R+Opportunistic pathogen associated with nosocomial infections
Enterobacter cancerogenus (9)99.11M, P, C, T, H+Associated with severe trauma or crush injuries
Enterococcus faecalis (2)99.78P, T+Nosocomial infections can cause endocarditis and bacteremia, urinary tract infections, meningitis, and other infections in humans
Enterococcus sulfureus (1)99.79NANot known
Escherichia hermannii (5)99.06C, T, P+Isolated from an infant patient with conjunctivitis, pathogenicity remains undetermined
Halomonas cupida (2)98.96NANot known
Holospora obtusa (2)83.08NANot known
Ignatzschineria larvae (2)99.40T+Isolated from the maggot-infested wound and diabetic foot ulcer in a human patient suffering from myiasis
Kerstersia gyiorum (3)99.9R, G+Isolated from human leg wounds
Klebsiella pneumoniae (8)99.80C, T, D, P+Respiratory and systemic infections
Kurthia gibsonii (2)99.35NANot known
Lactococcus garvieae (1)99.93T+Well-recognized fish pathogen and considered a rare pathogen with low virulence in humans
Lactococcus lactis (4)99.69NANot known
Morganella morganii (1)98.74R+Opportunistic pathogen, frequently involved in urinary tract infections
Myroides odoratimimus (2)99.58NASeptic shock, pneumonia, and soft tissue infection
Naxibacter varians (6)98.46NANot known
Paludibacterium yongneupense (4)96.79NANot known
Pantoea anthophila (3)99.86P, C+Not known
Parabacteroides distasonis (1)97.62NASplenic abscess in a sickle cell patient
Paraprevotella clara (1)89.27NANot known
Phascolarctobacterium faecium (1)96.11NANot known
Photobacterium damselae (20)99.79NARarely causes septicemia and wound infection in children
Plesiomonas shigelloides (6)99.89NAAn emerging pathogen, causing mainly intestinal diseases in humans
Proteus mirabilis (14)99.46R+, −Responsible for a variety of community- or hospital-acquired illnesses; urinary tract, wound, and bloodstream infections
Proteus mirabilis (2)99.08NA+, −
Providencia alcalifaciens (11)99.93G, P, H, R, D, T+Recognized pathogen causing food poisoning or traveler's diarrhea and gastroenteritis
Providencia alcalifaciens (4)97.38R, G+
Providencia rustigianii (22)99.69NAIsolated from human feces and can colonize the human intestine
Providencia stuartii (2)99.36R+Invasive pathogen commonly associated with urinary infections in patients with indwelling urinary catheters; meningitis
Pseudomonas corrugata (1)99.31P+Not known
Pseudomonas fragi (10)99.58NACan cause food spoilage (dairy products)
Pseudomonas mendocina (5)99.24P+Human cases of endocarditis, spondylodiscitis, and sepsis
Pseudomonas plecoglossicida (8)99.59NACausative agent of bacterial hemorrhagic ascites of ayu (fish pathogen)
Ralstonia pickettii (2)100NAAn infrequent invasive pathogen in healthy individuals
Serratia rubidaea (2)97.85P, D+Invasive opportunistic pathogen
Shewanella baltica (8)99.49NANot known
Shigella flexneri (7)99.87T, P, G+Causes an acute bloody diarrhea known as shigellosis or bacillary dysentery
Staphylococcus simiae (1)99.79NANot known
Staphylococcus warneri (2)100D,T+Primary bacteremia-causing agents among the pediatric population
Stenotrophomonas maltophilia (1)99.7P+Emerging opportunistic pathogen causing multiple types of infections in humans
Vagococcus carniphilus (2)97.82H+Not known
Wohlfahrtiimonas chitiniclastica (2)98.72NA+, −Bacteremia and fulminant sepsis in humans
Wohlfahrtiimonas chitiniclastica (5)99.07H, R, T+, −
  • G, garden; P, public park; D, dump area/garbage; T, public toilet; H, hospital; C, canteen/restaurant; M, mutton shop/market; R, house/human residence; NA, not applicable.

  • +, culture-dependent method; −, culture-independent method.

Phylogenetic reconstruction of 16S rRNA gene sequences

Phylogenetic reconstruction revealed that all OTUs/phylotypes from cultured isolates and clones clustered within six major phylogenetic clades of bacteria (where clades are the number of phyla observed that contained representative sequences; see Supporting Information, Figs S1 and S2). Within the phylum Gammaproteobacteria, phylotype R148 and C106 did not branch with their nearest cultured homologues Cronobacter sakazakii and Morganella morganii although blast analysis showed 90.31% and 98.74% sequence similarity, respectively. This suggests that these sequences may represent novel taxa due to low 16S rRNA gene sequence similarity and phylogenetic divergence from the nearest cultured homologues. Phylotype T97 showed 16S rRNA gene sequence homology of 99.40% with Ignatzschineria larvae, but phylogenetically these sequences formed a tight cluster with Ignatzschineria ureiclastica, a recently characterized bacterium isolated from the gastrointestinal tract of adult flesh flies (Diptera: Sarcophagidae; Gupta et al., 2011; Fig. S1). Phylotype H99 from the Firmicutes phylum showed 97.81% sequence similarity to Vagococcus carniphilus but displayed divergence (due to low 16S rRNA gene sequence similarity) and may represent novel species within this genus (Fig. S1). From the clone library, only one phylotype belonged to Alphaproteobacteria and showed low 16S rRNA gene sequence similarity (83.1%) to Holospora obtusa, an endonuclear symbiont of Paramecium caudatum. This sequence formed a stable monophyletic branch separated from remaining clades and supported by a bootstrap value of 100 (Fig. S2).

Biodiversity analyses

Good's coverage of sequences by culture-dependent and culture-independent methods indicates substantial coverage of the bacterial diversity and suggests that any new isolate and clone sequences have only a 7.85% and 3.76% chance, respectively, to fall in an unknown species or new OTUs. Shannon and Simpson diversity indices (which measure ecosystem biodiversity; Table ) indicated that a greater diversity of sequences was present in the clone library, where the phylotypes recovered by culture-independent methods exhibited greater divergence and diversity than the cultured counterpart. The rarefaction curve (Fig. ) indicated that diversity was sampled with good level of confidence, and the majority of OTUs in the sample were detected. We also observed a significant decrease (P = 0.05) in the rate of OTU detection with an increasing number of isolate/clone sequences. All values of richness indices, Chao1, ACE, and Jackknife (Table ), showed the estimated number of OTUs which were essentially near to the number of observed OTUs, confirming the conclusion from rarefaction analysis that our sampling of sequences had covered majority of OTUs.

Rarefaction curves of OTUs clustered at 97% similarity from cultured isolate and clone library sequences. The rarefaction curve is the number of sequences sampled for 0.03 distance representing the mean parameter value for cultured isolates and clone library gut homogenate sequences according to culture-dependent and culture-independent methods. Rarefaction curves were generated based on analyses performed in dotur using the furthest neighbor assignment algorithm.

View this table:

Biodiversity indices calculated for cultured isolates and clone library sequences using dotur

Biodiversity indices+
Number of sequences102213
Number of OTUs2831
Shannon index2.992.78
Simpson index0.050.08
Chao I31.533.8
Good's coverage92.15%96.24%
  • +, culture-dependent method; −, culture-independent method.

  • 97% similarity clusters have been considered as OTUs using dotur.


In the present study, we used culture-dependent and culture-independent sequence-based approaches to assess the microbial communities associated with the gut of house flies (M. domestica L.) collected from different sampling sites that humans occupy. This study demonstrated numerous bacterial phylotypes, including a large number of opportunistic and potential pathogenic species. High coverage values indicate that the bacterial community was covered effectively, and the majority of bacterial phylotypes were represented. However, we recognize that no complex microbial community has ever been sampled to completion, and these are probably low estimates. The detection of microbiota sequences could increase if pyrosequencing is used, allowing in-depth microbial diversity analysis. Both the high species richness and diversity of the bacterial community reflect the nature of house flies, including feeding (e.g. consumption of an indiscriminate diet) and reproductive behaviors (e.g. larval development in decaying organic matter; Greenberg, 1973; Graczyk et al., 2001; Moon, 2002).

Our survey revealed a prevalence of Proteobacteria, and primarily the class Gammaproteobacteria (Fig. ), which supports data from studies of bacterial communities in several other species of arthropods including: the honeybee Apis mellifera (Jeyaprakash et al., 2003); the deer tick, Ixodes scapularis (Benson et al., 2004); Culicoides sonorensis, an orbivirus vector (Campbell et al., 2004); the gypsy moth Lymantria dispar L. (Broderick et al., 2004); wild Culex quinquefasciatus mosquito midgut (Pidiyar et al., 2004); the ant lion, Myrmeleon mobilis (Dunn & Stabb, 2005); bee species (Apoidea) (Mohr & Tebbe, 2006); and Drosophila melanogaster (Corby-Harris et al., 2007; Cox & Gilmore, 2007). In comparison with other higher Diptera, recent studies from wild and laboratory-reared D. melanogaster (Cox & Gilmore, 2007) and natural populations of D. melanogaster (Corby-Harris et al., 2007) fruit flies showed a wide range of bacterial species from the Proteobacteria, Firmicutes, and Bacteroidetes phyla, which is in agreement with the data presented for wild caught house flies in this study. The Gammaproteobacteria were the most diverse group isolated from flies (fruit flies & house flies), and while the relative proportions of the various phylotypes found in the fruit flies differ from the relative proportions in house flies, similar bacterial genera and species were observed in both studies. The phylotype representatives found in both fly species (fruit flies & house flies), include species within the genera Enterobacter, Klebsiella, Pantoea, Serratia, Morganella, Pseudomonas, and Stenotrophomonas. A recent survey of the gut of the Mediterranean fruit fly, Ceratitis capitata, revealed a dominant community of the Enterobacteriaceae, with prominent species of Klebsiella, found in combination with Citrobacter freundii, Enterobacter spp., Pantoea spp., Pectobacterium spp., and Providencia stuartii and with a minor community of Pseudomonas spp. (Behar et al., 2008). Similarly, we found that the house fly community was dominated by Enterobacteriaceae, but additionally we found Alcaligenaceae,Xanthomonadaceae, Pseudomonadaceae, Moraxellaceae, Aeromonadaceae, Enterococcaceae, Bacillaceae, and Streptococcaceae and predominantly the genera Providencia, Proteus, Enterobacter, Klebsiella,Pseudomonas, and Wohlfahrtiimonas (Table ). Interestingly, many of these phylotypes were highly similar to the types of bacterial species normally found associated with humans, domestic animals, and plants, suggesting that house flies may mediate interactions between bacteria and alternative hosts.

Our survey also revealed many pathogenic species of bacteria, albeit less frequently distributed, that pose significant threats to human health. Diverse species of bacterial pathogens have been isolated from house flies in other parts of the world (Adeyemi & Dipeolu, 1984; Sulaiman et al., 2000; Förster et al., 2007; Nmorsi et al., 2007; Butler et al., 2010). For instance, we identified Klebsiella spp., which causes a wide array of infections in humans from septicemia to pneumonia. Several previous surveys have isolated Klebsiella spp. from the gut as well as from external surfaces of flies collected in hospital settings (Shooter & Waterworth, 1944; Greenberg, 1971; Adeyemi & Dipeolu, 1984; Fotedar et al., 1992). In addition, house flies have been implicated in the carriage and transmission of drug-resistant strains of Klebsiella in hospitals (Fotedar et al., 1992). We also identified Enterobacter spp., an opportunistic pathogen causing increasing concern with nosocomial infections in the United States (National Nosocomial Infections Surveillance System, 2003). Acinetobacter spp., Staphylococcus warneri, Stenotrophomonas maltophilia, Aeromonas veronii, Shigella flexneri, Pseudomonas mendocina, and Serratia rubidaea also were isolated, and these organisms are important as emerging opportunistic pathogen that causes sepsis, urinary tract infections, gastrointestinal diseases, and multiple infections in humans (Table ). Of particular public health importance was the presence of Shigella spp. in the house flies in our survey. This organism causes bacillary dysentery or shigellosis in man, with an estimated 160 million annual episodes and 1.1 million deaths, most of which are children under 5 years old in developing countries (Sansonetti, 2001). A previous study involving the association of house flies and Shigella spp. noted a correlation of fly activity and dysentery incidence as well as a marked reduction in disease cases when fly control was enacted (Levine & Levine, 1991). In the current study, Providencia spp., Proteus sp., and M. morganii were isolated with Providencia and Proteus as the dominant cultured representatives (Table ). Recently, it was shown that live bacteria of Providencia spp., Proteus spp., and M. morganii were found in the gut of newly emerged adult house flies and established that Providencia spp. and M. morganii could carry over in the gut from larval metamorphosis to adult eclosion (Su et al., 2010). As Providencia spp., Proteus spp., and M. morganii are responsible for a wide range of human infections (Table ), and Providencia spp. and Proteus spp. were dominantly represented in our survey, house flies can possibly serve as putative reservoirs or vectors for these microorganisms. In our clone library, the most dominant genus represented was Aeromonas, and Aeromonas hydrophila, an enteric pathogen (Efuntoye, 1996), was the dominant species. The genus Aeromonas has been linked with wide variety of human infections, acting as primary agent as well as opportunistic pathogen to immunocompromised patients. Several previous studies have isolated Aeromonas spp. from house flies (Gray et al., 1990; Nayduch et al., 2001; Rahuma et al., 2005). Aeromonas hydrophila, a causative agent of gastroenteritis, was predominantly isolated from the blow fly Chrysomya megacephala and also from M. domestica and Musca sorbens (Sulaiman et al., 2000). Our study supports the possibility that house flies may represent a potential hazard to public health, where they can serve as transporters, vectors, and reservoirs for human primary and opportunistic pathogens.

Diverse genera associated with house flies (e.g. Acinetobacter, Bacillus, Enterobacter, Proteus, Escherichia, Klebsiella, Providencia, Pseudomonas, Citrobacter, Micrococcus, Methylobacterium, Enterococcus, and Staphylococcus) have been isolated globally in several other studies (Sulaiman et al., 1988; Nazni et al., 2005; Förster et al., 2007; Nmorsi et al., 2007; Sukontason et al., 2007; Bouamamaa et al., 2010; Butler et al., 2010). Results from our culture-dependent method confirm data reported in these studies along with new reports of genera, and species therein described below, namely Kerstersia, Ignatzschineria, Wohlfahrtiimonas, Pantoea, Cronobacter, and Vagococcus. Kerstersia gyiorum is an Alcaligenes faecalis-like organism, and the majority of strains have been isolated from human leg wounds (Coenye et al., 2003). Ignatzschineria larvae was isolated previously from first and second larval stages of an obligate parasitic fly, Wohlfahrtia magnifica (Diptera: Sarcophagidae), a major myiasis-causing fly species (Tóth et al., 2001). No further reports on this bacterium in flies exist. Recently, we reported two novel species, Ignatzschineria indica and I. ureiclastica, isolated from gastrointestinal tract of adult flesh flies (Diptera: Sarcophagidae; Gupta et al., 2011). A recently described gammaproteobacterium, W. chitiniclastica, which was originally isolated from W. magnifica, can cause bacteremia in humans (Rebaudet et al., 2009) and belongs to a distinct lineage close to I. larvae (Tóth et al., 2008). We also found Pantoea anthophila in our survey, which has been previously isolated from flowering shrubs Impatiens balsamina in India (Brady et al., 2009). In a previous study, C. sakazakii was recovered via culture of collected house flies (Butler et al., 2010), and in our study, we isolated this bacterium from the house fly gut. Vagococcus carniphilus, a newly described species, has not been well studied, and its clinical significance, if any, has yet to be determined. The cloning and sequencing approach revealed the overall bacterial diversity (richness and abundance) and newly reported bacterial taxa in house flies such as Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, and Bacteroidetes and many new genera (Desulfovibrio, Holospora, Ralstonia, Comamonas, Naxibacter, Achromobacter, Paludibacterium, Wohlfahrtiimonas, Halomonas, Shewanella, Photobacterium, Plesiomonas, Clostridium, Kurthia, Phascolarctobacterium, Paraprevotella, Chryseobacterium, Myroides, Dysgonomonas, and Parabacteroides). Together, the results from our cultured and clone libraries suggest that house fly gut harbors a vast and previously uncovered bacterial diversity, which could not formerly be explored using only culture-based techniques.

Culture-based studies, which afford the opportunity to isolate viable organisms, are inherently useful for understanding the physiological, metabolic, and biochemical potential of isolated organisms. In addition, while culture-based studies can provide a good indication of ecosystem complexity, they do not necessarily provide comprehensive information on the composition of microbial communities as they are limited in scope to organisms that are permissive to cultivation on the utilized growth media and conditions. Some studies speculate that cultivation methods recover < 1% of the total microorganisms present in environmental samples (Staley & Konopka, 1985; Amann et al., 1995). In comparison, culture-independent studies allow for the identification of the cultivable and noncultivable fractions of microorganisms present in the sample and could cover most of the species present in the community. However, these molecular-based techniques require the user to standardize DNA extractions, PCR, and cloning conditions, so that bias will not be introduced due to primer selection or low representation of some genera or species in the community. Using both approaches along with taxonomy and phylogenetic techniques allows for better and more thorough characterization of microbial diversity.

The house fly gut represents an intriguing and unexplored niche for analyzing microbial ecology, which will provide opportunities for research involving the impact of diverse and dynamic microbial communities on human health and disease. This basic study revealed the microbial diversity that exists within the gut of adult house flies collected from different human environments by combining culture-based and molecular techniques. In this study, most of the bacterial species isolated from adult house flies have been documented to be either opportunistic pathogens or nonpathogens. Public awareness of the house fly as a reservoir and possibly a vector for these organisms should be tantamount, especially for children, the elderly, and the immunocompromised in the case of opportunistic pathogens. Future studies will address questions that could not be answered by sequencing phylogenetic markers. For example: what physiological functions, if any, are performed by resident bacteria in the house fly gut? What are the complex microbial-ecological events that govern the house fly–microorganism interactions in the alimentary canal? What factors are responsible for genetic variation in house fly-associated microorganisms? How do microorganisms survive and possibly develop resistance against antimicrobials?

Supporting Information

Additional Supporting Information may be found in the online version of the article:

Fig. S1. Unrooted phylogenetic dendogram of 16S rRNA genes amplified from cultured isolates from house fly gut homogenate, with representative sequences from the EzTaxon database.

Fig. S2. Unrooted phylogenetic dendogram of 16S rRNA genes amplified from clone library of house fly gut homogenate, with representative sequences from the EzTaxon database.

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We thank Dr G. C. Mishra, Director, National Centre for Cell Science (NCCS), Modern College, Pune, India, and Georgia Southern University, Statesboro, GA, USA, for facilities, encouragement, and support. Grants from Department of Biotechnology, New Delhi, supported this research. Research fellowship awarded by Council of Scientific and Industrial Research (CSIR), New Delhi, to A.K. Gupta, is gratefully acknowledged. We are grateful to Dr Rajnikant Dixit, National Institute of Malaria Research (NIMR, India), Dr Mahesh Dharne, Dr Ashraf Rangrez, and Mr C.P. Antony for providing valuable constructive ideas and comments in the initial stage of the study. Special thanks to Mrs Pragati Gupta for her helpful comments in preparing the manuscript.


  • Editor: Julian Marchesi


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