RESEARCH ARTICLE

Phylogenetic characterization of bacteria in the gut of house

flies (Musca domestica L.)
Arvind K. Gupta1, Dana Nayduch2, Pankaj Verma1, Bhavin Shah3, Hemant V. Ghate4, Milind S.
Patole1 & Yogesh S. Shouche1
1
Molecular Biology Unit, National Centre for Cell Science, Pune, Maharashtra, India; 2USDA-ARS, Center for Grain and Animal Health Research,
Arthropod-Borne Animal Diseases Research Unit, Manhattan, KS, USA; 3Department of Health Science, Pune University, Pune, Maharashtra, India;
and 4Department of Zoology, Modern College of Arts, Science and Commerce, Pune, Maharashtra, India

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Correspondence: Yogesh S. Shouche,
Molecular Biology Unit, National Centre for
Cell Science, Pune 411007, Maharashtra,
India. Tel.: +91 20 25708050;
fax: +91 20 25692259; e-mail:
yogesh@nccs.res.in
Received 12 June 2011; revised 24
September 2011; accepted 26 October 2011.
Final version published online 12 December
2011.
DOI: 10.1111/j.1574-6941.2011.01248.x
Editor: Julian Marchesi
MICROBIOLOGY ECOLOGY
Abstract
House flies (Musca domestica L.) are cosmopolitan, ubiquitous, synanthropic
insects that serve as mechanical or biological vectors for various microorgan-
isms. To fully assess the role of house flies in the epidemiology of human dis-
eases, 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 col-
lected 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 iso-
lates 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

Keywords
16S rRNA gene; culture dependent; clone
library; culture independent; bacterial
diversity.
construction of a 16S rRNA gene clone library, and sequence analyses sup-
ported culture recovery results. However, additional bacterial taxa not deter-
mined via culture recovery were revealed using this methodology and included
members of the classes Alphaproteobacteria, Deltaproteobacteria, and the phy-
lum 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.

mobile, they transport bacteria from septic environments

Introduction
Dipteran flies are one of the most abundant and impor-
tant 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 man-
ure, 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
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 vec-
tors 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

FEMS Microbiol Ecol 79 (2012) 581–593 ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
582
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 pseudo-
tuberculosis (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 stud-
ies. Historically, the first detailed observations of this nat-
ure were made by Graham-Smith (1910) who caught flies
randomly, artificially infected them with pathogenic bacte-
ria, 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 lim-
ited 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 sug-
gested that < 1% of bacterial species can be cultivated
(Staley & Konopka, 1985), and thus culture-dependent stud-
ies 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 asso-
ciated microbiota of house flies collected from various
public places that represented both sanitary and unsani-
tary areas. These sites included a garden, public park, gar-
bage 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
ª 2011 Federation of European Microbiological Societies
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A.K. Gupta et al.
utilizes 16S rRNA gene sequence analysis for surveying
house fly gut bacteria. The 16S rRNA gene sequences gen-
erated 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
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Sample collection
A total of 65 adult house flies were independently cap-
tured 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), hos-
pital (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 abun-
dance of adult house flies and existence of ecological con-
ditions 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 plas-
tic 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 ster-
ile 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 lL 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 cyto-
chrome oxidase I (COI) genes. Fly guts were sonicated
for 30 s, macerated with a plastic pestle, and then vor-
texed 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 col-
ony 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.
FEMS Microbiol Ecol 79 (2012) 581–593
Bacteria in the gut of house flies
Bacterial cultures from house fly gut
homogenate
A 100-lL aliquot of the gut contents was serially diluted
up to 10 6 and plated on Luria–Bertani (LB) agar, Tryp-
tic 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) glyc-
erol/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 bacte-
rial isolates from the house fly gut homogenate were sub-
cultured 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 (Sam-
brook et al., 1989) for PCR amplification. For culture-
independent identification of gut microbiota, total bacterial
community DNA was extracted from remaining gut
homogenate (~ 400 lL) using a DNeasy Blood & Tissue
Kit (Qiagen) with the following modifications to the
manufacturer’s protocol: (1) lysozyme (100 lL per sam-
ple, 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 lL
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 con-
centration of extracted DNA was checked on NanoDrop
ND-1000 spectrophotometer (NanoDrop Biotechnologies)
and ranged between 200 and 300 ng lL 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′)
FEMS Microbiol Ecol 79 (2012) 581–593
583
and 16R1525XP (5′-TTCTGCAGTCTAGAAGGAGGTG
WTCCAGCC-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-lL reaction mixture containing 200 lM (each)
dNTPs, 1 lL of a 10 lM each primer, 1 lL of (~ 50 ng)
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DNA template, and 2.5 U of Taq DNA polymerase (Ban-
galore Genei, India) with 19 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 homo-
genates 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 prod-
ucts into pGEM-T easy vector (Promega) according to
manufacturer’s instructions. Ligation mixture was trans-
formed in chemically competent E. coli JM109 cells with
30-min recovery time. Transformants were grown over-
night on LB plates containing 100 lg mL 1 each of
ampicillin, X-gal, and Isopropylb-D-1-thiogalactopyrano-
side. Two hundred and fifty white colonies were ran-
domly selected and screened for insert by PCR using
vector-specific M13F (5′-CGCCAGGGTTTTCCCAGT-
CACGAC-3′) and M13R (5′-TCACACAGGAAACAGC-
TATGAC-3′) primers. PCR was performed by incubating
the mixture at 94 °C for 7 min (to lyse the cells and ini-
tial 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.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
584
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 com-
pared 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 unambigu-
ous 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 gener-
ated 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 assess-
ment 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). Phylo-
genetic 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)] 9 100, 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 simi-
larity 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 rich-
ness and evenness; the Simpson Index [D = (Σn(n 1))/
ª 2011 Federation of European Microbiological Societies
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A.K. Gupta et al.
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 com-
pare 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
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been deposited in the GenBank database under accession
numbers HQ407007-HQ407321.
Results
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 cyto-
chrome oxidase I (COI) genes (Simon et al., 1994; Kraf-
sur 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 cul-
tured isolates and the clone library sequences, respec-
tively, 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 differ-
ent taxa is presented in Fig. 1. The majority of isolate
(89.21%) and clone (59.62%) sequences belonged to the
class Gammaproteobacteria. Isolates belonging to the clas-
ses Alphaproteobacteria, Deltaproteobacteria, and Bacteroi-
detes 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 1. We identified
seven novel phylotypes showing  97% sequence simi-
larity (Table 1) that could represent new genera or spe-
cies. A total of 41 different genera covering 55 different
phylotypes were recovered by applying both methods
(culture-dependent and culture-independent). Compara-
tive studies revealed 10 genera, and three species
(Wohlfahrtiimonas chitiniclastica, Acinetobacter soli, and
Proteus mirabilis) common in cultured isolates and the
clone library (Table 1). This indicates that the culture-
independent studies bolstered the cultured results with
many additional genera. Interestingly, 12 genera found in
FEMS Microbiol Ecol 79 (2012) 581–593
Bacteria in the gut of house flies
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 1).
These results imply that both culture-dependent and cul-
ture-independent methods are needed to unveil the total
microbial diversity of house fly gut.
Phylogenetic reconstruction of 16S rRNA gene
sequences
Phylogenetic reconstruction revealed that all OTUs/phylo-
types from cultured isolates and clones clustered within
six major phylogenetic clades of bacteria (where clades
are the number of phyla observed that contained repre-
sentative sequences; see Supporting Information, Figs S1
and S2). Within the phylum Gammaproteobacteria, phylo-
type 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. Phylo-
type 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 diver-
gence (due to low 16S rRNA gene sequence similarity)
Fig. 1. Percentage distribution of cultured isolates and clone library
sequences in different taxa based on 16S rRNA gene sequences.
FEMS Microbiol Ecol 79 (2012) 581–593
585
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 endo-
nuclear 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
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Good’s coverage of sequences by culture-dependent and
culture-independent methods indicates substantial cover-
age 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 2) indicated that a
greater diversity of sequences was present in the clone
library, where the phylotypes recovered by culture-inde-
pendent methods exhibited greater divergence and diver-
sity than the cultured counterpart. The rarefaction curve
(Fig. 2) indicated that diversity was sampled with good
level of confidence, and the majority of OTUs in the sam-
ple were detected. We also observed a significant decrease
(P = 0.05) in the rate of OTU detection with an increas-
ing number of isolate/clone sequences. All values of rich-
ness indices, Chao1, ACE, and Jackknife (Table 2),
showed the estimated number of OTUs which were essen-
tially near to the number of observed OTUs, confirming
the conclusion from rarefaction analysis that our sam-
pling of sequences had covered majority of OTUs.
Discussion
In the present study, we used culture-dependent and cul-
ture-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 phylo-
types 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.
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Published by Blackwell Publishing Ltd. All rights reserved
 Table 1. List of bacteria identified based on taxonomic assessment of 16S rRNA gene sequences from the EzTaxon database and their summary of medical significance
586

Homologue identified (abundance) Percent identity Collection site* Method† Medical significance

Achromobacter ruhlandii (1) 97.72 NA Not known

Acinetobacter bereziniae (2) 98.68 NA Acinetobacter spp. have been implicated in nosocomial infections
Acinetobacter haemolyticus (2) 99.62 M, R +
Acinetobacter radioresistens (1) 96.93 C +
Acinetobacter soli (1) 99.79 H +,
Acinetobacter soli (7) 97.88 NA +,
Aeromonas hydrophila (38) 99. 80 NA Gastroenteritis and wound infections
Aeromonas veronii (3) 100 P, T + Opportunistic gastrointestinal pathogen
Bacillus amyloliquefaciens (1) 99.57 H + Not known
Bacillus firmus (32) 99.44 NA Not known
Chryseobacterium haifense (1) 96.98 NA Not known
Clostridium sordellii (2) 98.56 NA Isolated from the immunocompromised patient’s stool

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ª 2011 Federation of European Microbiological Societies
Comamonas testosterone (4) 99.79 NA Rarely implicated as a human pathogen
Cronobacter sakazakii (1) 90.31 C + Recognized as causative agent of neonatal bacteraemia, meningitis, and necrotizing enterocolitis
Desulfovibrio senezii (18) 99.69 NA Not known
Dysgonomonas mossii (1) 99.17 NA Isolated from intestinal juice of a patient with pancreatic cancer
Enterobacter aerogenes (3) 97.11 G, R + Opportunistic pathogen associated with nosocomial infections
Enterobacter cancerogenus (9) 99.11 M, P, C, T, H + Associated with severe trauma or crush injuries
Enterococcus faecalis (2) 99.78 P, T + Nosocomial infections can cause endocarditis and bacteremia, urinary tract infections,
meningitis, and other infections in humans
Enterococcus sulfureus (1) 99.79 NA Not known
Escherichia hermannii (5) 99.06 C, T, P + Isolated from an infant patient with conjunctivitis, pathogenicity remains undetermined
Halomonas cupida (2) 98.96 NA Not known
Holospora obtusa (2) 83.08 NA Not known
Ignatzschineria larvae (2) 99.40 T + Isolated from the maggot-infested wound and diabetic foot ulcer in a human patient suffering
from myiasis
Kerstersia gyiorum (3) 99.9 R, G + Isolated from human leg wounds
Klebsiella pneumoniae (8) 99.80 C, T, D, P + Respiratory and systemic infections
Kurthia gibsonii (2) 99.35 NA Not known
Lactococcus garvieae (1) 99.93 T + Well-recognized fish pathogen and considered a rare pathogen with low virulence in humans
Lactococcus lactis (4) 99.69 NA Not known
Morganella morganii (1) 98.74 R + Opportunistic pathogen, frequently involved in urinary tract infections
Myroides odoratimimus (2) 99.58 NA Septic shock, pneumonia, and soft tissue infection
Naxibacter varians (6) 98.46 NA Not known
Paludibacterium yongneupense (4) 96.79 NA Not known
Pantoea anthophila (3) 99.86 P, C + Not known
Parabacteroides distasonis (1) 97.62 NA Splenic abscess in a sickle cell patient
Paraprevotella clara (1) 89.27 NA Not known
Phascolarctobacterium faecium (1) 96.11 NA Not known
Photobacterium damselae (20) 99.79 NA Rarely causes septicemia and wound infection in children
Plesiomonas shigelloides (6) 99.89 NA An emerging pathogen, causing mainly intestinal diseases in humans
A.K. Gupta et al.

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 Table 1. Continued
Bacteria in the gut of house flies

FEMS Microbiol Ecol 79 (2012) 581–593

Homologue identified (abundance) Percent identity Collection site* Method† Medical significance

Proteus mirabilis (14) 99.46 R +, Responsible for a variety of community- or hospital-acquired illnesses; urinary tract, wound,
and bloodstream infections
Proteus mirabilis (2) 99.08 NA +,
Providencia alcalifaciens (11) 99.93 G, P, H, R, D, T + Recognized pathogen causing food poisoning or traveler’s diarrhea and gastroenteritis
Providencia alcalifaciens (4) 97.38 R, G +
Providencia rustigianii (22) 99.69 NA Isolated from human feces and can colonize the human intestine
Providencia stuartii (2) 99.36 R + Invasive pathogen commonly associated with urinary infections in patients with indwelling
urinary catheters; meningitis
Pseudomonas corrugata (1) 99.31 P + Not known
Pseudomonas fragi (10) 99.58 NA Can cause food spoilage (dairy products)
Pseudomonas mendocina (5) 99.24 P + Human cases of endocarditis, spondylodiscitis, and sepsis
Pseudomonas plecoglossicida (8) 99.59 NA Causative agent of bacterial hemorrhagic ascites of ayu (fish pathogen)
Ralstonia pickettii (2) 100 NA An infrequent invasive pathogen in healthy individuals
Serratia rubidaea (2) 97.85 P, D + Invasive opportunistic pathogen
Shewanella baltica (8) 99.49 NA Not known
Shigella flexneri (7) 99.87 T, P, G + Causes an acute bloody diarrhea known as shigellosis or bacillary dysentery
Staphylococcus simiae (1) 99.79 NA Not known
Staphylococcus warneri (2) 100 D,T + Primary bacteremia-causing agents among the pediatric population
Stenotrophomonas maltophilia (1) 99.7 P + Emerging opportunistic pathogen causing multiple types of infections in humans
Vagococcus carniphilus (2) 97.82 H + Not known
Wohlfahrtiimonas chitiniclastica (2) 98.72 NA +, Bacteremia and fulminant sepsis in humans
Wohlfahrtiimonas chitiniclastica (5) 99.07 H, 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.

ª 2011 Federation of European Microbiological Societies

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587

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588 A.K. Gupta et al.

Table 2. Biodiversity indices calculated for cultured isolates and clone recent studies from wild and laboratory-reared D. mela-
library sequences using DOTUR nogaster (Cox & Gilmore, 2007) and natural populations
of D. melanogaster (Corby-Harris et al., 2007) fruit flies
Biodiversity indices + showed a wide range of bacterial species from the Proteo-
Number of sequences 102 213
bacteria, Firmicutes, and Bacteroidetes phyla, which is in
Number of OTUs* 28 31 agreement with the data presented for wild caught house
Singletons 8 8 flies in this study. The Gammaproteobacteria were the
Shannon index 2.99 2.78 most diverse group isolated from flies (fruit flies & house
Simpson index 0.05 0.08 flies), and while the relative proportions of the various
Chao I 31.5 33.8 phylotypes found in the fruit flies differ from the relative
ACE 34.48 38.31

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Jackknife 36 39
Good’s coverage 92.15% 96.24%
+, culture-dependent method; , culture-independent method.
*97% similarity clusters have been considered as OTUs using DOTUR.
Fig. 2. 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.
larval development in decaying organic matter; Green-
berg, 1973; Graczyk et al., 2001; Moon, 2002).
Our survey revealed a prevalence of Proteobacteria, and
primarily the class Gammaproteobacteria (Fig. 1), which
supports data from studies of bacterial communities in
several other species of arthropods including: the honey-
bee Apis mellifera (Jeyaprakash et al., 2003); the deer tick,
Ixodes scapularis (Benson et al., 2004); Culicoides sonoren-
sis, 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,
proportions in house flies, similar bacterial genera and
species were observed in both studies. The phylotype rep-
resentatives found in both fly species (fruit flies & house
flies), include species within the genera Enterobacter, Kle-
bsiella, Pantoea, Serratia, Morganella, Pseudomonas, and
Stenotrophomonas. A recent survey of the gut of the Medi-
terranean fruit fly, Ceratitis capitata, revealed a dominant
community of the Enterobacteriaceae, with prominent
species of Klebsiella, found in combination with Citrob-
acter freundii, Enterobacter spp., Pantoea spp., Pectobacte-
rium 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, Pseudomonad-
aceae, Moraxellaceae, Aeromonadaceae, Enterococcaceae,
Bacillaceae, and Streptococcaceae and predominantly the
genera Providencia, Proteus, Enterobacter, Klebsiella, Pseu-
domonas, and Wohlfahrtiimonas (Table 1). 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 signif-
icant 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; But-
ler 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 exter-
nal surfaces of flies collected in hospital settings (Shooter
& Waterworth, 1944; Greenberg, 1971; Adeyemi & Dipeo-
lu, 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 Nos-
ocomial Infections Surveillance System, 2003). Acinetobacter

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 79 (2012) 581–593
Published by Blackwell Publishing Ltd. All rights reserved
Bacteria in the gut of house flies
spp., Staphylococcus warneri, Stenotrophomonas maltophil-
ia, 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, gas-
trointestinal diseases, and multiple infections in humans
(Table 1). 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 dis-
ease 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 Pro-
teus as the dominant cultured representatives (Table 1).
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 1), 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 Aeromo-
nas 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 trans-
porters, vectors, and reservoirs for human primary and
opportunistic pathogens.
Diverse genera associated with house flies (e.g. Acineto-
bacter, Bacillus, Enterobacter, Proteus, Escherichia, Klebsiella,
Providencia, Pseudomonas, Citrobacter, Micrococcus, Meth-
ylobacterium, 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.,
FEMS Microbiol Ecol 79 (2012) 581–593
589
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, Wohl-
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fahrtia magnifica (Diptera: Sarcophagidae), a major myia-
sis-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. chitiniclas-
tica, 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, Wohlfahrtii-
monas, Halomonas, Shewanella, Photobacterium, Plesio-
monas, Clostridium, Kurthia, Phascolarctobacterium,
Paraprevotella, Chryseobacterium, Myroides, Dysgonomon-
as, and Parabacteroides). Together, the results from our
cultured and clone libraries suggest that house fly gut
harbors a vast and previously uncovered bacterial diver-
sity, 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 under-
standing the physiological, metabolic, and biochemical
potential of isolated organisms. In addition, while cul-
ture-based studies can provide a good indication of eco-
system complexity, they do not necessarily provide
comprehensive information on the composition of micro-
bial communities as they are limited in scope to organ-
isms that are permissive to cultivation on the utilized
growth media and conditions. Some studies speculate that
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
590
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 intro-
duced 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 charac-
terization of microbial diversity.
The house fly gut represents an intriguing and unex-
plored 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 combin-
ing 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 ques-
tions that could not be answered by sequencing phyloge-
netic 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?
Acknowledgements
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 Sci-
entific 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.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
A.K. Gupta et al.
Special thanks to Mrs Pragati Gupta for her helpful com-
ments in preparing the manuscript.
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Supporting Information
Additional Supporting Information may be found in the
online version of this 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
Downloaded from https://academic.oup.com/femsec/article-abstract/79/3/581/491160 by guest on 21 August 2019
genes amplified from clone library of house fly gut
homogenate, with representative sequences from the
EzTaxon database.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials sup-
plied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
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Published by Blackwell Publishing Ltd. All rights reserved