International Journal of Medical Microbiology 306 (2016) 566–571

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International Journal of Medical Microbiology

journal homepage: www.elsevier.com/locate/ijmm

A geospatial analysis of flies and the spread of antimicrobial resistant

bacteria
Frieder Schaumburg a,∗ , Francis Chinedum Onwugamba a , Ruslan Akulenko b ,
Georg Peters a , Alexander Mellmann c , Robin Köck a,1 , Karsten Becker a
a
Institute of Medical Microbiology, University Hospital Münster, Domagkstr. 10, 48149 Münster, Germany
b
Center for Bioinformatics, University of Saarland, Campus E2 1, 66041 Saarbrücken, Germany
c
Institute of Hygiene, University Hospital Münster, Robert-Koch-Str. 41, 48149 Münster, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Livestock is often colonized with ESBL-producing Enterobacteriaceae (ESBL-E) and Staphylococcus aureus.
Received 30 March 2016 There is a risk that flies spread antimicrobial resistant bacteria from livestock to humans. Here, we
Received in revised form 30 May 2016 screened flies from urban and rural areas near the city of Münster, Germany, for the presence of ESBL-E
Accepted 12 June 2016
and S. aureus and compared molecular characteristics of these isolates with those isolated from humans
in the same region.
Keywords:
In total, 1346 single flies were grinded and cultured overnight in BHI-broth. The broth was cultured
Diptera
on Columbia blood agar and selective media for the detection of S. aureus and ESBL-E. Human isolates
Staphylococcus aureus
Enterobacteriaceae
from routine diagnostics at the University Hospital Münster were used for comparison. Antimicrobial
Extended-spectrum beta-lactamase susceptibility, phylogroups (Escherichia coli), spa types/multilocus sequence types (S. aureus) and selected
Methicillin-resistance antimicrobial resistance genes were determined for each isolate. GPS data of the sampling sites were used
to map flies carrying ESBL-E and S. aureus.
Overall, Serratia fonticola (123/1346; 9.1%) was the most prevalent ESBL-E in flies, followed by E. coli
(44/1346; 3.3%). A high proportion of flies was positive for ESBL-producing E. coli (15.0–22.2%) in a
rural area. CTX-M-1 was the most prevalent beta-lactamase in E. coli (38.6%). One livestock-associated
methicillin resistant S. aureus (LA-MRSA, t011/ST398) was found in the city centre of Münster. Overall, a
substantial part of ESBL-producing E. coli and S. aureus from flies and humans showed a similar genetic
background.
In conclusion, flies can carry ESBL-E and LA-MRSA in urban and rural areas. The similar genetic back-
ground of isolates from humans and flies points towards a common source.
© 2016 Elsevier GmbH. All rights reserved.

1. Introduction In Germany, livestock-associated MRSA (LA-MRSA) mostly

Infections caused by methicillin-resistant Staphylococcus aureus
(MRSA), extended-spectrum beta-lactamase- and carbapenemase-
producing Enterobacteriaceae (ESBL-E, CP-E) are amongst the major
challenges in human healthcare. Huge efforts were undertaken to
prevent the spread of these “super bugs”. However, effective infec-
tion control is hampered by reservoirs that are difficult to reach.
For instance, livestock, such as pigs, is frequently colonized with
ESBL-E (30.2%) and MRSA (20.7%) (Schmithausen et al., 2015).
∗ Corresponding author.
E-mail address: frieder.schaumburg@ukmuenster.de (F. Schaumburg).
1
Present address: Institute of Hospital Hygiene Oldenburg, Medical Campus, Uni-
versity of Oldenburg, Oldenburg, Germany.
belong to multilocus sequence typing (MLST) clonal complex 398
(CC 398), which has crossed the species barrier causing up to one
third of all human MRSA infection/colonization in some regions
(Köck et al., 2013; van Alen et al., 2016). While direct contact
to livestock is the major risk factor to acquire LA-MRSA, inhala-
tion of contaminated dust can increase LA-MRSA colonization rates
in exposed personnel (Bos et al., 2016). Colonization of livestock
with ESBL-E can be associated with ESBL-E colonization in humans
pointing towards a direct transmission (Dohmen et al., 2015;
Kluytmans et al., 2013). Recent studies reported ESBL-E on flies
in the vicinity of livestock farms in the Netherlands and Germany
(Blaak et al., 2014; von Salviati et al., 2015). Colonization of flies
with ESBL-E might promote the spread of antibiotic resistant bac-
teria from farms to urban places. This could be feasible as flight

http://dx.doi.org/10.1016/j.ijmm.2016.06.002
1438-4221/© 2016 Elsevier GmbH. All rights reserved.
 F. Schaumburg et al. / International Journal of Medical Microbiology 306 (2016) 566–571
distance of house flies (Musca domestica) ranges between 5 and
7 km.
The Northwestern part of Germany (including the area around
the city of Münster) has the highest density of pigs (between 600
and >900 pigs/ha) and cattle (150 and >200 cattle/ha, http://www.
atlas-agrarstatistik.nrw.de) in Germany. Our hypothesis was that
flies ingest ESBL-E and S. aureus in the livestock environment and
disperse it even in urban areas.
The objective of the study was to screen flies from the city of
Münster and the rural region in its surrounding for ESBL-E and S.
aureus and to compare these isolates with ESBL-E and S. aureus
isolated from humans.
2. Materials and methods
2.1. Ethical approval
Ethical approval to report patient-related data was granted by
the Ethical Committee of the University of Münster (2016-008-f-S).
2.2. Study area
The study area comprised urban (city of Münster) and rural areas
(villages, farmland) around Münster (Fig. 1). Münster has approxi-
mately 300,000 inhabitants and is the cultural capital of the region
of Westphalia in Northwestern Germany. The North-South exten-
sion of the study area was 15 km, the East-West extension was
18 km resulting in an overall size of approximately 270 km2 (Fig. 1).
2.3. Flies
Flies (Diptera) were collected between April and July 2015 in a
gaze trap placed over a bait containing proteins, lipids and minerals
(Feldner, Waldsee, Germany). Flies were killed in 50 ml tubes filled
with 10 ml 70% ethanol. Ethanol sanitizes the body surface of the
flies to rule out cross-contamination in the trap without killing the
intestinal microbiome (Gupta et al., 2012). Not only the exoskeleton
but also more importantly the gastrointestinal tract is the source
of pathogen transmission from flies to humans (i.e. regurgitation,
faecal deposition on food items) (Graczyk et al., 2001).
Flies were dried on silica gel (2–5 mm, Carl Roth, Karlsruhe,
Germany) to remove the ethanol off the exoskeleton and were
cultured within 2 h. Species identification of flies was done phe-
notypically.
Single flies were grinded with a sterile pestle in 1.5 ml tubes
and incubated in 1 ml BHI-broth overnight at 35 ◦ C. 10 ␮l of the
broth suspension were cultured on Columbia blood agar and selec-
tive media for the detection of S. aureus (SAID, bioMérieux, Marcy
l’Etoile, France) and ESBL-E (chromID, bioMérieux).
For each sampling point, environmental data (i.e. sky cover,
presence of refuse dump, decomposing organic matters, livestock
faeces in a 10 m radius), the setting (urban, rural) and weather
conditions (temperature, humidity, windforce, air pressure, sun-
shine hours/day, as reported by the “Deutscher Wetterdienst” for
the region Münster/Osnabrück) were recorded.
2.4. Bacterial isolates from humans
Bacterial isolates from humans were consecutively collected
in the routine microbiology laboratory of the University Hospi-
tal Münster (January–May 2015). All isolates were recovered from
clinical and screening specimens of hospital in- and outpatients.
Only one isolate per patient was included. All patient-related data
(date of birth, sex, date of admission, infection/colonization) were
extracted from the laboratory software.
567
2.5. Identification
Species identification of isolates from flies and human was
done using MALDI-TOF (microflex LT, Bruker Daltonics, Bremen,
Germany). Species confirmation of Enterobacteriaceae was done
using VITEK2 automated systems (bioMérieux). Species of S. aureus
was confirmed by the PCR-detection of the S. aureus specific ther-
mostable nuclease nuc (Brakstad et al., 1992).
2.6. Antimicrobial resistance
Antimicrobial susceptibility testing was done with VITEK2 auto-
mated systems (bioMérieux) using EUCAST clinical breakpoints.
The ESBL-phenotype in Enterobacteriaceae was confirmed by the
double disk diffusion test (Mast discs, Mast Diagnostics, Boo-
tle, UK). In addition, ESBL-E were screened for the presence of
blaCTX-M , blaSHV , blaTEM and blaCMY-2 (Monstein et al., 2007; Souna
et al., 2014). PCR amplicons were sequenced to type the respective
bla gene. Carbapenem resistance was assessed by the isothermal
amplification of genes encoding KPC, VIM, NDM, and OXA-48- and
OXA-181-group carbapenemases (eazyplex SuperBug CRE, Amplex,
Gießen, Germany).
The methicillin-resistance in S. aureus was confirmed by PCR
targeting mecA (Murakami et al., 1991).
2.7. Genotyping
ESBL-producing Escherichia coli were phylogrouped follow-
ing the revised scheme by Clermont et al. (Clermont et al.,
2013). Presence of clonal group A among isolates belong-
ing to phylogroup D was tested using a PCR-based assay
(Johnson et al., 2004). A minimum spanning tree was con-
structed for all ESBL-producing E. coli using SeqSphere+ (Ridom,
Münster, Germany) based on categorical data derived from resis-
tance against piperacillin/tazobactam, cefotaxime, ceftazidime,
ertapenem, ciprofloxacin, trimethoprim/sulfamethoxazole, CTX-
M- and TEM-type as well as E. coli phylogroups.
All S. aureus were spa typed and one isolate per spa type of each
group (flies, humans) was randomly selected for MLST (Enright
et al., 2000; Mellmann et al., 2006).
2.8. Mapping
GPS data from all sampling points were collected and the
proportion of ESBL-E colonized flies at each sampling point was
mapped to “Google maps” (https://maps.google.de) using “R” sta-
tistical software and the “plotGoogleMaps” (version 2.2) package.
2.9. Statistical analysis
Categorical variables were compared with chi2 or Fisher’s exact
test and the Odds Ratio was calculated to assess the strength of
association. The significance level was set at 0.05. Analyses were
done with “R” statistical software.
3. Results
3.1. Sampling
In total, 1346 flies including Musca domestica (n = 895), Cal-
liphora sp. (n = 447) and others (n = 4) were collected at 80 sampling
sites (Fig. 1).
The sampling sites were rural (n = 32, 40%) or urban (n = 48, 60%).
At some sampling sites, refuse dumps (n = 22, 27.5%), decompos-
ing organic matters (n = 34, 42.5%) and animal faeces (n = 17, 21.2%)
were seen within a 10 m radius around the flytrap. A mean number
568 F. Schaumburg et al. / International Journal of Medical Microbiology 306 (2016) 566–571
Fig. 1. Map of sampling sites where flies with ESBL-producing E. coli were found in the city of Münster and surrounding region. Each circle represents one of the 80 sampling
sites. The size/colour corresponds to the proportion of flies colonized with ESBL-producing E. coli. A mean number (±SD) of 16.8 (±5.0) flies per site were trapped. The black
border line encircles the urban area.
(±SD) of 16.8 (±5.0) flies were caught with a median (range) of 16
(3–80) flies per hour.
The traps were set at 41 days; the mean temperature (±SD) was
16.3 ◦ C (±4.7), the mean relative humidity (±SD) reached 61.6%
(±7.1), the median (range) wind force came to 2 Bft. (1–4) and the
mean (±SD) air pressure was 1013.0 hPa (±6.4). The median (range)
sunshine hours during the 41 days were 9.0 h (1.0–15.4).
All isolates growing on ESBL-selective media were confirmed
to produce ESBL by double-disk diffusion test. In total, ESBL-E were
found in flies at 62 (77.5%) sites. The most prevalent ESBL-E (n = 176)
was Serratia fonticola (n = 123), followed by E. coli (n = 44), Rahnella
aquatilis (n = 5) and Klebsiella pneumoniae, K. oxytoca, Pantoea sp.,
Proteus mirabilis (n = 1, each). In total, five S. aureus were found at
five sampling sites.
Among the three most abundant species, only ESBL-producing
E. coli and S. aureus are of medical importance and were there-
fore compared with consecutively collected isolates from humans.
ESBL-producing E. coli and S. aureus from 93 and 82 patients, respec-
tively, were included (Table 1). While ESBL-producing E. coli was
isolated in similar proportions from colonization and infection, the
majority of S. aureus derived from infections (i.e. skin, soft tissue
and bone).
3.2. ESBL-producing E. coli in flies and humans
In general, 3.3% (n = 44) of all flies were positive for ESBL-
producing E. coli. Isolates from flies were less resistant to
piperacillin/tazobactam, ceftazidime, ciprofloxacin and trimetho-
prim/sulfamethoxazole compared to isolates from humans
(Table 2). Two ESBL-producing E. coli from humans were resistant
to ertapenem and intermediate to meropenem. One of these was
resistant and one was susceptible to imipenem. A porin loss was
most likely the underlying mechanism of carbapenem resistance.
Genes encoding KPC, VIM, NDM or OXA like carbapenemases were
not detected.
CTX-M was predominant in ESBL-producing E. coli from humans
and flies (86% and 77.3%, respectively), followed by TEM (31.2%
and 25%, respectively) and SHV (2.2% and 0%, respectively). Only
one ESBL-producing E. coli (CTX-M-15 positive) from humans was
CMY-2 positive. While CTX-M-1 was associated with flies, CTX-
M-27 was only found in E. coli from humans (Table 2). Almost all
phylogroups were found in isolates from flies and humans but in
different proportions: E. coli phylogroup A was detected twice as
often in flies as in humans (79.6% vs. 32.3%, Table 2). In contrast,
phylogroups B2 and C were only found among E. coli from humans.
Clonal group A was not detected among isolates belonging to phy-
logroup D. Based on the susceptibility data, resistance genes and
phylogroups reported in Table 2, we constructed a minimum span-
ning tree to assess the relatedness of ESBL-producing E. coli from
flies and humans. Three clusters were found, one of them (clus-
ter 1) showed an almost equal distribution between isolates from
humans and flies (Fig. 2). Two nodes represented isolates from both
humans and flies. Closely related isolates from humans and flies
predominantly belonged to phylogroup A.
Mapping of the sampling sites revealed an increased proportion
(15–22.2%) of flies being positive for ESBL-producing E. coli in the
rural Northwestern part of the study region (Fig. 1).
3.3. Other ESBL-producers in flies
ESBL-producing S. fonticola from flies (n = 123) carried CTX-M
(n = 45, 36.6%) or TEM (n = 2, 1.6%). SHV or CMY-2 was not detected.
ESBL-producing S. fonticola were found all over the study area with-
out significant clusters. Of the five ESBL-producing R. aquatilis, four
carried CTX-M. The beta-lactamases TEM, SHV or CMY-2 were not
detected in R. aquatilis.
3.4. S. aureus in flies and humans
S. aureus was found in five flies collected at five different (6.3%)
sites. Four isolates were methicillin susceptible, and belonged to
spa types t091, t362, t1535 and t2985. Of these, two were tested
 F. Schaumburg et al. / International Journal of Medical Microbiology 306 (2016) 566–571 569

Table 1
Characterization of patients from whom ESBL-producing E. coli or S. aureus were isolated.

Infected/colonized patients

ESBL-producing E. coli S. aureus

Total 93 82
Mean age, years, (±SD) 54.7 (±27.3) 48.0 (±27.4)
Sex, female (n, %) 37 (39.8%) 37 (45.1%)
Community associateda (n, %) 45 (48.4%) 66 (80.5%)
Hospital associated (n, %) 48 (51.6%) 16 (19.5%)
Colonization (n, %) 50 (53.8%) 1 (1.2%)
Infection Total (n, %) 43 (46.2%) 81 (98.8%)
Urinary tract (n, %) 26 (28.0%) 7 (8.5%)
Abscess (n, %) 2 (2.2%) 0 (%)
Skin (n, %) 8 (8.6%) 38 (46.3%)
Soft tissue and bone (n, %) 1 (1.1%) 10 (12.2%)
Other (n, %) 7 (16.3%) 26 (31.7%)
a
If samples were taken within ≤48 h after admission, including patients attending the outpatient department.

Table 2
Comparison of ESBL-producing E. coli from flies and humans, Germany, 2015.

Flies (n = 44), n (%) Humans (n = 93), n (%) OR (95% CI) p-value

Antimicrobial Piperacillin/tazobactam 4 (9.1%) 67 (72%) 25 (7.8–100) <0.005

resistancea Cefotaxime 44 (100%) 90 (96.8%) inf (0.2–inf) 0.6
Ceftazidime 18 (40.9%) 75 (80.6%) 5.9 (2.6–14.3) <0.005
Ertapenem 0 (0%) 2 (2.2%) – –
Ciprofloxacin 18 (40.9%) 70 (75.3%) 4.3 (1.9–10) <0.005
Trimethoprim/sulfamethoxazole 10 (22.7%) 57 (61.3%) 5.2 (2.4–12.5) <0.005

Beta-lactamasesb CTX-M-1 17 (38.6%) 19 (20.4%) 0.3 (0.1–0.8) 0.007

CTX-M-15 9 (20.5%) 33 (35.5%) 2.0 (0.8–5.5) 0.12
CTX-M-27 0 (0%) 11 (11.8%) inf (1.2–inf) 0.02
CTX-M−9 groupc 10 (22.7%) 15 (16.1%) 0.7 (0.2–2.4) 0.6
TEM-1 10 (22.7%) 27 (29.0%) 2.7 (0.03–219.2) 0.5

Phylogroup A 35 (79.6%) 30 (32.3%) 0.1 (0.1–0.3) <0.005

B1 1 (2.3%) 3 (3.2%) 1.4 (0.1–14.2) 1
B2 0 (0%) 26 (28.0%) – –
C 0 (0%) 9 (9.7%) – –
D 3 (6.8%) 3 (3.2%) 0.5 (0.1–2.4) 0.4
E 1 (2.3%) 3 (3.2%) 1.4 (0.1–14.2) 1
F 3 (6.8%) 1 (1.1%) 0.2 (0.01–1.5) 0.1
Otherd 1 (2.3%) 18 (19.4%) 10.3 (1.3–80.0) 0.007
a
Includes resistant and intermediate isolates.
b
Isolates were only screened for the most common beta-lactamases. Therefore, the beta-lactamase genes do not add to the total number of isolates.
c
Excluding CTX-M-27.
d
Including clade I (n = 1), cryptic clade (n = 2) and isolates (“unknown”, n = 16) which were not assigned to any group based on the quadruplex-PCR by Clermont et al.
(2013).

penicillin susceptible. One MRSA was found in one fly trapped in the
city centre of Münster (51◦ 57 34 N, 7◦ 37 13 E). This MRSA showed
characteristics of LA-MRSA (t011, ST398), harboured mecA and was
resistant to trimethoprim/sulfamethoxazole and tetracycline but
was susceptible to aminoglycosides, quinolones and macrolides. All
S. aureus from flies and humans were susceptible to glycopeptides,
daptomycin and linezolid. Noteworthy, ST398-MRSA indicative for
LA-MRSA were predominant among all MRSA from humans (n = 8,
40%).
In general, resistance rates were similar in S. aureus from flies
and humans (Table 3). However, an in-depth statistical comparison
was omitted due to the small sample size of isolates from flies.
4. Discussion
We screened 1346 flies from the city of Münster and its sur-
rounding areas for the presence of ESBL-E and S. aureus and
compared molecular characteristics of these isolates with isolates
from humans. Main findings are a high proportion of flies colonized
with ESBL-producing E. coli particularly in a rural setting and the
detection of LA-MRSA in a fly in the city centre of Münster. A simi-
lar genetic background of isolates from flies and humans suggest a
common source.
The high proportion of S. fonticola among all ESBL-E is not sur-
prising as it is frequently encountered in the environment. For
instance, S. fonticola was the most abundant species (62.9%) among
ESBL-producers isolated from retail vegetables (van Hoek et al.,
2015). However, E. coli was the most frequent pathogen amongst
clinically important ESBL-producers in flies, while K. pneumoniae
was detected only once. This is in line with the general observation
that ESBL-producing E. coli is rather community-associated while
ESBL-producing Klebsiella sp. is mainly nosocomial-associated
(Pitout and Laupland, 2008).
ESBL-producing E. coli from flies were less resistant to
piperacillin/tazobactam, ceftazidime, ertapenem and ciprofloxacin
than isolates from humans (Table 2). Similarly, lower resistance
rates to piperacillin/tazobactam, ceftazidime, and ciprofloxacin
were found in ESBL-producers from meat products compared to
isolates from humans (Schaumburg et al., 2014).
CTX-M-1 was the most prevalent beta-lactamase in isolates
from flies, which is in line with an overall higher proportion of
the CTX-M-1 group in livestock compared to humans (Strauß et al.,
2015; von Salviati et al., 2015). In contrast, the CTX-M-9 group (incl.
CTX-M-27) is reported to be less frequent in livestock and the farm
environment, similar to our findings (Table 1) (von Salviati et al.,
2015). The non-detection of phylogroup B2 among E. coli from flies
570 F. Schaumburg et al. / International Journal of Medical Microbiology 306 (2016) 566–571

Fig. 2. Minimum spanning tree of ESBL-producing Escherichia coli from humans and flies. The tree was constructed based on resistance data against piperacillin/tazobactam,
cefotaxime, ceftazidime, ertapenem, ciprofloxacin, trimethoprim/sulfamethoxazole, CTX-M- and TEM-type as well as E. coli phylogroups (Table 2). Nodes are labelled with
E. coli phylogroups. Isolates from flies (white) and humans (grey) share cluster 1, 2 and 3. NK: not known (see Table 2).

Table 3
Comparison of S. aureus from flies and humans, Germany, 2015.

Flies (n = 5) Humans (n = 82)

Antimicrobial Penicillin 3 (60%) 53 (64.6%)

resistance, n (%) Cefoxitin 1 (20%) 20 (24.4%)
Erythromycin 1 (20%) 20 (24.4%)
Clindamycin 1 (20%) 19 (23.2%)
Gentamicin 0 (%) 3 (3.7%)
Trimethoprim/sulfamethoxazole 1 (20%) 4 (4.9%)

spa types MRSA (ST; n) t011 (ST398; 1) t003 (ST225; 2), t011 (ST398; 5), t022 (ST22; 1), t032
(ST22; 5), t034 (ST398; 3), t422 (1), t728 (ST45; 1), t1139
(ST22; 1), t2461 (ST72; 1)
MSSA (n) t091 (1), t362 (1), t1535 (1), t2985 (1) t002 (4), t005 (1), t008 (1), t012 (2), t015 (2), t018 (1), t021
(2), t026 (1), t037 (1), t050 (1), t064 (1), t073 (1), t078 (1),
t084 (4), t085 (1), t091 (8), t127 (3), t130 (1), t136 (1), t156
(1), t159 (1), t390 (1), t521 (1), t548 (1), t550 (1), t571 (1),
t587 (1), t608 (1), t728 (2), t1580 (1), t1639 (1), t1875 (1),
t2667 (1), t3537 (1), t15206 (2), t15207 (1), t15598 (1),
t15599 (1), t15600 (1), t15671 (1), non-typable (2)

can be expected as this phylogroup is rather associated with mam-
mals (Gordon et al., 2008). Albeit being less common, phylogroup
D and F were the second most frequent type in E. coli from flies
(Table 2). Phylogroup D was associated with more invasive isolates
in recent studies (Gordon et al., 2008).
Data from phenotypic susceptibility testing, resistance genes
and phylogroups were used to construct a minimum spanning tree
in order to assess the relatedness of E. coli from flies and humans.
We found that isolates from flies and humans were grouped in all
clusters (Fig. 2). This argues for a common third source of ESBL-
E. coli or a transmission of E. coli between flies and humans. In
general, flies can be a vector of resistant bacteria: A study from the
Netherlands analysed 87 pooled flies from a broiler and laying-hen
farm. Two out of 19 pools revealed the presence of ESBL-producing
E. coli (Blaak et al., 2014). E. coli from flies shared identical sequence
types with isolates from rinse water and manure in this study. Simi-
larly, a study from Czech Republic found 18% of flies being colonized
with ESBL-producing E. coli (Dolejska et al., 2011). The authors
concluded that isolates and resistance genes can be transmitted
between horses, flies and humans (Dolejska et al., 2011).
Although ESBL-producing E. coli was found throughout the
whole study region, a cluster of high colonization rates in flies
was identified in a rural area North-West of the city of Mün-
ster. As livestock and the farm environment can be a reservoir
of ESBL-producers, it is possible that livestock production could
be one source for the detection of ESBL-producers in flies (Blaak
et al., 2014; Schmithausen et al., 2015). However, we did not find
increased levels of ESBL-producing E. coli in flies in other rural areas
with a high density of livestock.
Although we detected CTX-M genes in S. fonticola and R. aquatilis
using validated protocols, there is a risk of misidentification. CTX-M
is closely related to the beta-lactamases FONA and SFO of S. fonticola
or RAHN of R. aquatilis and might have been therefore misdetected
by the applied primers (Bellais et al., 2001; Doublet et al., 2010).
Our study suggests that the intestinal MRSA colonization of flies
is rare in rural and urban settings. However, we provide a “proof of
 F. Schaumburg et al. / International Journal of Medical Microbiology 306 (2016) 566–571
principle” that flies can carry LA-MRSA even in urban settings. Since
we only analysed the intestinal colonization of flies, the overall col-
onization with S. aureus/MRSA might be underreported. One study
from Germany showed that the colonization of the exoskeleton
with S. aureus is higher (8.9%) than the detected intestinal colo-
nization rate in our study (0.4%, Table 3) (Förster et al., 2007). The
area surrounding Münster is a hotspot for CC398 LA-MRSA and their
introduction in the human healthcare system has led to a propor-
tion of approximately 20% LA-MRSA of all MRSA in this area (Köck
et al., 2013). Thus, livestock farming could be assumed to be the
most likely source of this LA-MRSA isolate but we cannot rule out
other sources. Humans as a source of LA-MRSA are rather unlikely as
the colonization rate of MRSA in humans in our study region is 0.7%
(Köck et al., 2016). Noteworthy, LA-MRSA from flies and humans in
our study shared the identical spa type (t011) pointing towards a
common source. To the best of our knowledge it is unknown if and
to what extent flies play a role in the spread of MRSA. Humans
might get infected from flies through the ingestion of food items
contaminated with faeces from flies. Here, the infectious dose may
be relevant when assessing the risk of MRSA transmission from flies
in future studies.
Few limitations of our study need to be addressed: First, the
minimum spanning tree based on phenotypic and genotypic data
has not been comprehensively validated with multilocus sequence
typing data. Therefore, only the relation of the isolates and not
the phylogenetic evolution can be deduced from our analysis. Sec-
ond, the cross-sectional design of our study does not allow for an
assessment of the direction of transmission. This can only be done
in prospective studies. Third, the isolates included from humans
were derived from hospital in- and outpatients and are therefore
not representative for isolates from the general population.
5. Conclusion
Flies can carry ESBL-E and LA-MRSA in urban and rural areas in
Germany. A similar genetic background of isolates from flies and
humans points towards a common source.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
We thank Fabian Leendertz for providing the custom-made fly-
trap. This study was supported by institutional funds and in part
by the German Federal Ministry of Education and Research (BMBF,
MedVet-Staph; 01KI1301A).
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