LETTER
Though 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 plasmid-mediated 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).
TABLE 1.
Plasmid-mediated quinolone resistance genes in Enterobacteriaceae from American crowsa
| Gene | No. of samples from: |
Total (%) (n = 590) (365 [61.9]; 192 [32.5]) | |||
|---|---|---|---|---|---|
| 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]) | ||
| qnrA | 3 | 3 (0.5) | |||
| qnrB2 | 1 | 1 (0.2) | |||
| qnrB4 | 1 | 1 (0.2) | |||
| qnrB5 | 1 | 1 (0.2) | |||
| qnrB6 | 7 | 6 | 13 | 1 | 27 (4.6) |
| qnrB9 | 1 | 2 | 7 | 1 | 11 (1.9) |
| qnrB10 | 8 | 3 | 9 | 3 | 23 (3.9) |
| qnrB12 | 1 | 1 (0.2) | |||
| qnrB13 | 1 | 2 | 2 | 1 | 6 (1.0) |
| qnrB16 | 1 | 1 (0.2) | |||
| qnrB17 | 3 | 3 | 6 (1.0) | ||
| qnrB18 | 1 | 1 (0.2) | |||
| qnrB19 | 1 | 1 | 5 | 7 (1.2) | |
| qnrB20 | 1 | 1 (0.2) | |||
| qnrB27 | 1 | 1 (0.2) | |||
| qnrB28 | 1 | 2 | 3 (0.5) | ||
| qnrB30 | 1 | 1 (0.2) | |||
| qnrB32 | 2 | 1 | 3 (0.5) | ||
| qnrB36 | 4 | 4 (0.7) | |||
| qnrB47 | 2 | 10 | 11 | 5 | 28 (4.7) |
| qnrB50 | 4 | 1 | 5 (0.8) | ||
| qnrB61 | 1 | 1 | 1 | 3 (0.5) | |
| qnrB64 | 1 | 1 (0.2) | |||
| qnrB65 | 1 | 1 (0.2) | |||
| qnrB66 | 1 | 1 (0.2) | |||
| qnrB67 | 2 | 1 | 3 (0.5) | ||
| qnrB68 | 3 | 1 | 4 (0.7) | ||
| qnrB69 | 1 | 2 | 3 (0.5) | ||
| qnrB70 | 1 | 1 (0.2) | |||
| qnrB71 | 1 | 1 (0.2) | |||
| qnrC | 0 (0) | ||||
| qnrD | 2 | 2 | 4b (0.7) | ||
| qnrS1 | 1 | 2 | 1 | 4 (0.7) | |
| qnrS2 | 2 | 2 (0.3) | |||
| aac(6′)-Ib-cr | 1 | 15 | 1 | 17 (2.9) | |
| oqxAB | 8 | 3 | 1 | 12 (2.0) | |
| qepA | 0 (0) | ||||
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).
To our knowledge, this is the first report of qnrD from the United States.
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 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.
TABLE 2.
Novel qnrB variants detected in Enterobacteriaceae from American crows
| Novel qnrB variant | GenBank accession no. | Bacterial origin | No. of samples | Collection site |
|---|---|---|---|---|
| 64 | KC580653 | Citrobacter freundii | 1 | California |
| 65 | KC580654 | Citrobacter freundii | 1 | Massachusetts |
| 66 | KC580655 | Citrobacter freundii | 1 | New York |
| 67 | KC580656 | Citrobacter braakii | 2 | Massachusetts |
| 1 | New York | |||
| 68 | KC580657 | Citrobacter braakii | 3 | California |
| 1 | Kansas | |||
| 69 | KC580658 | Citrobacter freundii | 1 | California |
| 2 | Kansas | |||
| 70 | KC580659 | Citrobacter braakii | 1 | Kansas |
| 71 | KC580660 | Citrobacter braakii | 1 | New York |
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 Regional 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.
Footnotes
Published ahead of print 18 November 2013
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