Plasmid-Mediated Quinolone Resistance
Genes in Enterobacteriaceae from
American Crows: High Prevalence of
Bacteria with Variable qnrB Genes
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Dana Halová, Ivo Papousek, Ivana Jamborova, Martina
Masarikova, Alois Cizek, Nicol Janecko, Veronika Oravcova,
Ludek Zurek, Anne B. Clark, Andrea Townsend, Julie C.
Ellis and Ivan Literak
Antimicrob. Agents Chemother. 2014, 58(2):1257. DOI:
10.1128/AAC.01849-13.
Published Ahead of Print 18 November 2013.
LETTER TO THE EDITOR
Plasmid-Mediated Quinolone Resistance Genes in Enterobacteriaceae
from American Crows: High Prevalence of Bacteria with Variable qnrB
Genes
Dana Halová,a Ivo Papoušek,a Ivana Jamborova,a Martina Masarikova,b,c Alois Cizek,b,c Nicol Janecko,a,d Veronika Oravcova,a,c
Ludek Zurek,e Anne B. Clark,f Andrea Townsend,g Julie C. Ellis,h Ivan Literaka,c
T
hough wild birds are not normally exposed to use of antimicrobial agents, they can acquire antibiotic-resistant bacteria
through the environment (1). It was also suggested that rooks may
disseminate these bacteria over long distances and pose a risk of
contaminating the environment (2). A recent study of rooks (Corvus frugilegus) revealed that the frequencies of ciprofloxacin-resistant Enterobacteriaceae range from 3% to 92% in Europe. The
objective of this study was to describe the occurrence of plasmidmediated quinolone resistance (PMQR) genes in the Enterobacteriaceae of American crows (Corvus brachyrhynchos), which are the
North American ecological equivalent to the rooks in Eurasia.
Fresh crows’ feces were collected as described previously (2) in
four states throughout the United States in 2012. Swabs were cultivated in buffered peptone water overnight and subcultivated on
MacConkey agar (MCA) supplemented with ciprofloxacin (0.05
mg/liter). Pooled DNA was extracted from different colonies on
MCA and tested by PCR for the PMQR genes aac(6=)-Ib, qepA,
qnrA, qnrB, qnrC, qnrD, qnrS, and oqxAB (2–5), which were subsequently sequenced. Matrix-assisted laser desorption ionization–
time of flight mass spectrometry determined the bacterial strains
that had new variants of PMQR genes.
Enterobacteriaceae bacteria resistant to ciprofloxacin were isolated from 62% (365/590) of samples. The prevalence varied from
43% (California) to 81% (New York). PMQR genes were detected
in 33% (192/590) of samples (Table 1).
The level of resistant bacteria observed in wild animals is often
positively correlated with the degree of association with humans (6).
However, with the increased effort to limit the use of fluoroquinolones in food production in the United States (http://www.fda.gov/
AnimalVeterinary/SafetyHealth/RecallsWithdrawals/ucm042004
.htm; accessed May 2013), we might expect lower prevalence. Our
numbers of samples with resistant bacteria and with detected PMQR
genes are significantly higher than those in an equivalent European
study (2).
One possible explanation is that crows are exposed to resistant
bacteria of human origin. Other factors to explain the PMQR
prevalence variation include the proximity of study sites to urban
areas, density of human populations, and variation in levels of
environmental contamination. However, as some American crow
populations are migratory, the PMQR resistance prevalence may
not correlate directly with the level of antibiotic use in the immediate vicinity of the sampled regions (1).
Within the PMQR isolates, there was a wide range of qnr genes
February 2014 Volume 58 Number 2
TABLE 1 Plasmid-mediated quinolone resistance genes in
Enterobacteriaceae from American crowsa
No. of samples from:
Gene
qnrA
qnrB2
qnrB4
qnrB5
qnrB6
qnrB9
qnrB10
qnrB12
qnrB13
qnrB16
qnrB17
qnrB18
qnrB19
qnrB20
qnrB27
qnrB28
qnrB30
qnrB32
qnrB36
qnrB47
qnrB50
qnrB61
qnrB64
qnrB65
qnrB66
qnrB67
qnrB68
qnrB69
qnrB70
qnrB71
qnrC
qnrD
qnrS1
qnrS2
aac(6=)-Ib-cr
oqxAB
qepA
California
(n ⫽ 198)
(86 [43.4];
37 [18.7])
Kansas
(n ⫽ 149)
(111 [74.5];
48 [32.2])
Massachusetts
(n ⫽ 200)
(133 [66.5];
83 [41.5])
New York
(n ⫽ 43)
(35 [81.4];
24 [55.8])
3
1
1
7
1
8
6
2
3
1
2
1
1
13
7
9
2
1
3
1
1
3
1
3
1
1
1
5
1
1
1
2
1
4
10
1
1
2
4
1
1
2
1
11
5
1
1
2
3
1
1
1
1
2
1
1
2
2
1
1
8
2
1
2
15
3
1
1
Total (%)
(n ⫽ 590)
(365 [61.9];
192 [32.5])
3 (0.5)
1 (0.2)
1 (0.2)
1 (0.2)
27 (4.6)
11 (1.9)
23 (3.9)
1 (0.2)
6 (1.0)
1 (0.2)
6 (1.0)
1 (0.2)
7 (1.2)
1 (0.2)
1 (0.2)
3 (0.5)
1 (0.2)
3 (0.5)
4 (0.7)
28 (4.7)
5 (0.8)
3 (0.5)
1 (0.2)
1 (0.2)
1 (0.2)
3 (0.5)
4 (0.7)
3 (0.5)
1 (0.2)
1 (0.2)
0 (0)
4b (0.7)
4 (0.7)
2 (0.3)
17 (2.9)
12 (2.0)
0 (0)
a
Parenthetical values in the column headings are as follows: (number of samples
collected [n]) (number [percent] of Enterobacteriaceae bacteria resistant to
ciprofloxacin; number [percent] with PMQR genes).
b
To our knowledge, this is the first report of qnrD from the United States.
Published ahead of print 18 November 2013
Address correspondence to Dana Halová, [email protected].
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01849-13
Antimicrobial Agents and Chemotherapy
p. 1257–1258
aac.asm.org
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Downloaded from http://aac.asm.org/ on March 6, 2014 by Alois Cizek
Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech
Republica; Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech
Republicb; CEITEC VFU, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republicc; Department of Population Medicine, University of Guelph,
Guelph, Canadad; Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USAe; Department
of Biological Sciences, Binghamton University, Binghamton, New York, USAf; Department of Wildlife, Fish, & Conservation Biology, University of California at Davis, Davis,
California, USAg; Cummings School of Veterinary Medicine, Tufts University, North Grafton, Massachusetts, USAh
Letter to the Editor
TABLE 2 Novel qnrB variants detected in Enterobacteriaceae from
American crows
GenBank
accession no.
Bacterial origin
64
65
66
67
KC580653
KC580654
KC580655
KC580656
Citrobacter freundii
Citrobacter freundii
Citrobacter freundii
Citrobacter braakii
68
KC580657
Citrobacter braakii
69
KC580658
Citrobacter freundii
70
71
KC580659
KC580660
Citrobacter braakii
Citrobacter braakii
No. of
samples
Collection site
1
1
1
2
1
3
1
1
2
1
1
California
Massachusetts
New York
Massachusetts
New York
California
Kansas
California
Kansas
Kansas
New York
found. The most predominant gene was qnrB, which was detected
in 25% of samples, and the variants qnrB6, qnrB10, and qnrB47
were the most common. In contrast, qnrS1 was the most predominant subtype in recent reports (5, 7). Eight novel variants of qnrB
(qnrB64, qnrB65, qnrB66, qnrB67, qnrB68, qnrB69, qnrB70, and
qnrB71) were described in Citrobacter spp. (Table 2). The genes
aac(6=)-Ib-cr, oqxAB, qnrD, qnrS1, qnrA1, and qnrS2 were found
in 17, 12, 4, 4, 3, and 2 samples, respectively. qepA and qnrC genes
were not detected. Nineteen samples with more than one resistance gene were found, with a combination of qnrB and aac(6=)Ib-cr being most common.
Additional studies are needed to determine where crows acquire resistant bacteria, how long they persist in the gastrointestinal tracts of the birds, and whether they can potentially be transmitted to humans.
Nucleotide sequence accession numbers. Sequences of the
novel qnrB variants have been deposited in GenBank under accession numbers KC580653 to KC580660.
ACKNOWLEDGMENTS
This study was funded by the Central European Institute of Technology
(CEITEC) project (grant CZ.1.05/1.1.00/02.0068) of the European Re-
1258
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Antimicrobial Agents and Chemotherapy
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Novel qnrB
variant
gional Development Fund and by the Education for Competitiveness Operational Programme project (grant CZ.1.07/2.3.00/30.0014) of the European Social Fund.
We thank Mária Mičudová, Raluca Uricariu, Kateřina Albrechtová,
Monika Dolejska, Marie Slavíková, Jana Hofírková, Jarmila Kovářová,
and Eva Suchanová for excellent cooperation in the laboratory.