Antimicrobial Susceptibility Profiles and Molecular Typing of Campylobacter jejuni and Campylobacter coli Isolates from Ducks in South Korea Bai Wei, Se-Yeoun Cha, Min Kang, Jae-Hee Roh, Hye-Suk Seo, Ran-Hee Yoon, Hyung-Kwan Jang

Campylobacter is a food-borne zoonotic pathogen that causes human gastroenteritis worldwide. Campylobacter bacteria are commensal in the intestines of many food production animals, including ducks and chickens. The objective of the study was to determine the prevalence of Campylobacter species in domestic ducks, and the agar dilution method was used to determine resistance of the isolates to eight antibiotics. In addition, multilocus sequence typing (MLST) was performed to determine the sequence types (STs) of selected Campylobacter isolates. Between May and September 2012, 58 duck farms were analyzed, and 56 (96.6%) were positive for Campylobacter. Among the isolates, 82.1% were Campylobacter jejuni, 16.1% were C. coli, and one was unidentified by PCR. Of the 46 C. jejuni isolates, 87.0%, 10.9%, and 21.7% were resistant to ciprofloxacin, erythromycin, and azithromycin, respectively. Among the C. coli isolates, all 9 strains were resistant to ampicillin, and 77.8% and 33.3% were resistant to ciprofloxacin and azithromycin, respectively. The majority of the Campylobacter isolates were classified as multidrug resistant. Twenty-eight STs were identified, including 20 STs for C. jejuni and 8 STs for C. coli. The most common clonal complexes in C. jejuni were the ST-21 complex and the ST-45 complex, while the ST-828 complex predominated in C. coli. The majority of isolates were of STs noted in ducks and humans from earlier studies, along with seven STs previously associated only with human disease. These STs overlapped between duck and human isolates, indicating that Campylobacter isolates from ducks should be considered potential sources of human infection.

C

ampylobacters are the most commonly isolated bacterial enteric pathogens in developed and developing countries (1). Campylobacter jejuni is the predominant cause of campylobacteriosis, which is responsible for 93.4% of confirmed cases, whereas C. coli accounts for only 2.3% of outbreaks (2). Acute diarrheal illness is the main clinical sign, but more severe complications, such as Guillain-Barré syndrome, reactive arthritis, and a range of extraintestinal infections, occur in some serious cases (3, 4). Macrolides and fluoroquinolones are normally considered for treatment of Campylobacter enteritis; however, the prevalence of fluoroquinolone-resistant Campylobacter has increased dramatically in many countries since the last century, and such strains continue to exist with high-level resistance (5–7). Poultry is the major source of human infection; chickens constitute the major reservoir of Campylobacter, highlighting the potential public health threat (8, 9). Ducks are the second largest poultry meat reservoir with a potential public health risk, but they have remained largely uncharacterized in comparison to chickens. There is some evidence of an increased risk of Campylobacter infection in humans associated with ducks (10). A study in the United Kingdom found that duck meat was implicated in 2% of campylobacteriosis outbreaks (11), and a large outbreak of campylobacteriosis was derived from duck products in the United Kingdom (12). Previous surveys indicated high prevalences of Campylobacter in domestic ducks and duck meat (11, 13). Therefore, there is reason to suspect that ducks are an important source of human campylobacteriosis that may be underestimated. An increase in Campylobacter resistance, particularly to fluoroquinolones and macrolides, has also been found in ducks (14–16). These findings indicate the potential risk of antibiotic-resistant Campylobacter subsequently being transferred to humans by ducks.

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The proportion of global duck meat production occurring in Asia increased from 79.0% to 82.9% between 2002 and 2011 (17). In South Korea, duck production represents an important sector of animal husbandry, and consumption of duck meat has increased significantly, at an average rate of 13.4%, since 2001, which is faster than the case for any other meat source (18). However, little information is available on the prevalence and antimicrobial resistance of Campylobacter strains from ducks. Therefore, we investigated the occurrence and antimicrobial resistance of Campylobacter in domestic ducks in South Korea. In addition, we characterized antimicrobial resistance and correlated it with genotype. MATERIALS AND METHODS Isolation and identification of Campylobacter. From May to September 2012, 58 farms were selected from five provinces. Five, 6, 12, 15, and 20 farms in Gyonggi, Chungnam, Chungbuk, Chonnam, and Chonbuk Provinces (Fig. 1), respectively, were sampled, which included ⬎90% of the duck population in the five provinces (19). The sizes of the different farms varied from 5,000 to 20,000 ducks; the ducks were reared indoors with low curtain walls but not a closed production system until 6 to 7 weeks of age. Among the 58 farms, 9, 12, 19, and 18 farms had ducks of less

Received 25 July 2014 Accepted 23 September 2014 Published ahead of print 26 September 2014 Editor: M. W. Griffiths Address correspondence to Hyung-Kwan Jang, [email protected]. B.W. and S.-Y.C. contributed equally to this article. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.02469-14

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Departments of Infectious Diseases & Avian Diseases, College of Veterinary Medicine and Korea Zoonosis Research Institute, Chonbuk National University, Iksan, South Korea

Campylobacter spp. Isolated from Ducks in South Korea

RESULTS FIG 1 Locations of provinces in South Korea and prevalences of Campylobacter isolated in different provinces. The numbers represent the numbers of positive farms/total numbers of farms.

than 1 week of age, less than 2 weeks but more than 1 week of age, less than 3 weeks but more than 2 weeks of age, and more than 3 weeks of age, respectively. Five pooled cloacal swabs from each farm were preenriched in Bolton broth (Oxoid Ltd., Basingstoke, England) and supplemented with cefoperazone, vancomycin, trimethoprim, and cycloheximide (Oxoid) for 24 h at 42°C in a microaerobic atmosphere of 10% CO2, 5% O2, and 85% N2. The bacteria were streaked onto modified charcoalcefoperazone-desoxycholate agar (Oxoid) containing an antibiotic supplement (cefoperazone and amphotericin; Oxoid) and incubated for 48 h at 42°C under microaerobic conditions. Presumptive Campylobacter colonies on the plate were further cultivated on 5% sheep blood agar plates (Komed, Seongnam, South Korea) for 24 to 48 h at 42°C under microaerobic conditions. One suspected colony was isolated from each farm. DNA templates were prepared using freshly grown Campylobacter colonies on blood agar by adding 500 ␮l sterile distilled water and boiling the samples in a heater block at 100°C for 15 min. Template DNA was stored at ⫺20°C until it was used for PCR. The isolates were identified to the genus level by amplifying the 16S rRNA gene specific for Campylobacter species (20). We used multiplex PCR with specific primers to identify Campylobacter species, the hipO gene specific for C. jejuni, and the glyA gene specific for C. coli (21). After identifying each isolate, the Campylobacter isolates were initially stored in 20% glycerol in brain heart infusion broth (Oxoid) at ⫺70°C. Antimicrobial susceptibility testing. The agar dilution method was used to determine susceptibility of the Campylobacter isolates to the following eight antimicrobial agents: ampicillin (Amp), azithromycin (Azi), ciprofloxacin (Cip), clindamycin (Cli), erythromycin (Ery), gentamicin (Gen), nalidixic acid (Nal), and tetracycline (Tet). Mueller-Hinton agar (Oxoid) plates supplemented with 5% lysed sheep blood (Oxoid) were used as culture medium for 2-fold serial dilutions of antibiotics ranging from 0.06 to 256 ␮g/ml. Fresh bacterial colonies taken directly from the agar plates and incubated for 24 h were resuspended in sterile tryptic soy broth (Merck Inc., Rahway, NJ) to obtain a suspension with a 0.5 McFarland turbidity. The plates were incubated under microaerobic conditions at 42°C for 24 h. MIC values were defined as the lowest concentrations producing no visible growth, and antimicrobial-free agar plates were included as a control for normal growth. The breakpoints were deter-

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Isolation and identification of Campylobacter. Of the 58 farms, 56 (96.6%) had Campylobacter. Among the 56 isolates, 82.1% (46/56 isolates), 16.1% (9/56 isolates), and 1.8% (1/56 isolates) were identified as C. jejuni, C. coli, and unidentified Campylobacter species, respectively. The isolation rates were 95.0% (19/20 farms) in Chonbuk, 93.3% (14/15 farms) in Chonnam, 100.0% (12/12 farms) in Chungbuk, 100.0% (6/6 farms) in Chungnam, and 100.0% (5/5 farms) in Gyonggi (Fig. 1). All five provinces had high rates of Campylobacter isolation, with no significant differences (P ⬎ 0.05). All 9 C. coli isolates were isolated from Chungbuk Province, with a higher prevalence (75.0% [9/12 farms]) than that of C. jejuni (25.0% [3/12 farms]). Except for one farm with ducks of ⬍1 week of age and one farm with ducks of ⬎3 weeks of age which were negative for Campylobacter, Campylobacter was successfully isolated from ducks of the other age groups. Antimicrobial susceptibility. The antimicrobial susceptibilities of the 46 C. jejuni and 9 C. coli strains were determined against eight antimicrobial agents, and the results are presented in Table 1. Among the C. jejuni isolates, resistance to ciprofloxacin was the most common (40/46 isolates [87.0%]), followed by resistance to tetracycline (39/46 isolates [84.8%]) and nalidixic acid (39/46 isolates [84.8%]). All C. coli strains were resistant to ampicillin, and 88.9% (8/9 isolates) of the isolates were resistant to tetracycline. Bacterial resistance to macrolides (azithromycin and erythromycin) was also determined and presented resistance rates of 21.7% and 33.3%, respectively, for C. jejuni and 33.3% and 33.3%, respectively, for C. coli. Except for 1 strain of C. jejuni which was susceptible to all eight antimicrobial agents, the other 55 strains of Campylobacter were resistant to at least one antimicrobial agent (Table 2). The results indicate that 92.7% (51/55 isolates) of the strains were resistant to at least two antimicrobial agents, and the most frequent multidrug resistance pattern was resistance to ampicillin, ciprofloxacin, nalidixic acid, and tetracycline. All C. coli isolates were multidrug resistant. One isolate of C. jejuni and one isolate of C. coli showed resistance to all eight antimicrobial agents tested in this study. MLST. Twenty-eight different STs were identified among the samples: 20 STs for C. jejuni and 8 STs for C. coli (Table 3). Three complexes, namely, the ST-21 complex, the ST-45 complex, and the ST-828 complex, predominated and accounted for 60% of all

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mined according to the National Antimicrobial Resistance Monitoring System criteria (22). Since there were no ampicillin breakpoints for Campylobacter, we used the breakpoints for Enterobacteriaceae from the Clinical and Laboratory Standards Institute criteria (23). C. jejuni ATCC 33560 was used as the reference quality control strain. MLST. All C. jejuni and C. coli isolates were characterized by multilocus sequence typing (MLST) based on primers for seven gene targets of each isolate, including aspA (encoding aspartase A), glnA (encoding glutamine synthase), gltA (encoding citrate synthase), glyA (encoding serine hydroxymethyl transferase), pgm (encoding phosphoglucomutase), tkt (encoding transketolase), and uncA (encoding the ATP synthase alpha subunit), using previously described conditions (24). Allele numbers, sequence types (STs), and clonal complexes (CCs) were assigned using the PubMLST database (http://pubmlst.org/campylobacter/) (25), and novel STs were submitted to the MLST database and assigned new numbers. Statistical analysis. The chi-square test was used for comparisons in order to determine if there were statistically significant differences at the 95% level in the prevalence of Campylobacter samples between different provinces. Differences were considered significant if the P values were ⬍0.05.

Wei et al.

TABLE 1 Distribution of MICs of eight antimicrobial agents for C. jejuni and C. coli isolates No. of isolates for each concn of antimicrobial (␮g/ml) ⱕ0.06

0.13

0.25

0.5

1

2

4

8

16

32

64

128

ⱖ256

Breakpoint (␮g/ml)

MIC50/MIC90 (␮g/ml)

Resistance rate (%)

Ampicillin

C. jejuni C. coli

0 0

0 0

0 0

0 0

0 0

3 0

2 0

4 0

7 0

8 5

3 0

2 1

16 4

32 32

32/ⱖ256 32/ⱖ256

64.4 100.0

Azithromycin

C. jejuni C. coli

11 3

7 0

5 1

5 3

4 0

0 0

3 0

3 0

2 2

1 0

4 1

8 8

0.25/32 0.5/16

22.2 30.0

Ciprofloxacin

C. jejuni C. coli

2 0

1 0

1 1

0 0

0 1

2 0

1 1

2 0

7 5

20 2

9 0

4 4

32/64 16/32

86.7 80.0

Clindamycin

C. jejuni C. coli

2 0

8 1

10 1

12 3

4 1

3 3

3 0

1 0

2 1

0 0

0 0

8 8

0.5/4 0.5/2

6.7 10.0

Erythromycin

C. jejuni C. coli

2 0

8 2

8 1

8 3

5 0

4 0

4 1

1 0

0 0

0 0

1 1

4 2

32 32

0.5/64 0.5/128

11.1 30.0

Gentamicin

C. jejuni C. coli

5 0

4 1

10 0

11 6

10 2

0 0

1 0

1 0

0 0

0 0

0 0

3 1

8 8

0.5/4 0.5/1

8.9 10.0

Nalidixic acid

C. jejuni C. coli

0 0

0 0

3 1

1 0

1 1

2 1

2 1

19 4

17 2

64 64

128/ⱖ256 128/ⱖ256

84.4 80.0

Tetracycline

C. jejuni C. coli

1 0

4 0

2 0

0 1

1 0

6 3

10 0

4 0

17 6

16 16

64/ⱖ256 256/ⱖ256

84.4 90.0

TABLE 2 Antimicrobial resistance patterns of C. jejuni and C. coli isolates No. of agents to Campylobacter which isolate(s) Antimicrobial resistance species is resistant profile C. jejuni

1

2 3

4 5 6

8

C. coli

2 4 6 8

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No. of Rate isolates (%)

Cip Nal Tet Amp Tet Cip Nal Amp Azi Tet Amp Cip Nal Amp Cip Tet Amp EryTet Cip Nal Tet Amp Azi Cip Nal Amp Cip Nal Tet Amp Azi Cip Nal Tet Amp Cip Gen Nal Tet Amp Azi Cip Ery Nal Tet Amp Azi Cip Gen Nal Tet Amp Cip Cli Gen Nal Tet Azi Cip Cli Ery Nal Tet Amp Azi Cip Cli Ery Gen Nal Tet

1 1 1 1 1 1 2 1 1 10 1 13 3 1 2 1 1 1 1

2.2 2.2 2.2 2.2 2.2 2.2 4.4 2.2 2.2 22.2 2.2 28.9 6.7 2.2 4.4 2.2 2.2 2.2 2.2

Amp Cip Amp Tet Amp Azi Ery Tet Amp Cip Nal Tet Amp Azi Cip Ery Nal Tet Amp Azi Cip Cli Ery Gen Nal Tet

1 1 1 5 1 1

10.0 10.0 10.0 50.0 10.0 10.0

isolates. The most common of these was the ST-21 complex, with 17 isolates divided into 3 STs, followed by the ST-45 complex, represented by 8 isolates in 2 STs, and the ST-828 complex, with 8 isolates and 7 STs. Forty-four of the isolates grouped into seven previously characterized CCs, whereas the remaining nine isolates had STs that were unassigned. Three new STs were identified in this study, but no new allelic sequences were found for any of the housekeeping genes, and all of the new STs resulted from new combinations of previously described alleles. A total of 73 alleles were identified across all seven loci, ranging from 7 alleles of glyA to 15 alleles of gltA. DISCUSSION

Ducks and their products are commonly consumed in the modern Asian diet, but little information is available about Campylobacter species from ducks. Therefore, the objective of this study was to determine the prevalence of Campylobacter infection in ducks from South Korea. Campylobacter was isolated from 96.6% of the duck cloacal samples, and such a high isolation rate of Campylobacter from ducks has been reported previously (13, 16). The present isolation rate was much higher than that from chicken flocks in China and Japan, with isolation rates of 77.7% and 47.2%, respectively (26, 27). Differences in avian species, temperature, moisture, and feed model may have influenced the prevalence of Campylobacter (28, 29). C. jejuni is typically identified as the most prevalent species on commercial duck farms among the scarce Campylobacter studies on domestic ducks (13, 14), and our results were consistent with these findings, as 82.1% of the isolates were identified as C. jejuni. We found a high prevalence (88.9%) of Campylobacter in ducks of ⬍1 week of age. A similar result has been reported previously, with 100% of ducks being contaminated with Campylobacter at 8 days of age (30). This result was different from the case for chickens, in

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Species

Antimicrobial agent

Campylobacter spp. Isolated from Ducks in South Korea

TABLE 3 Sources and STs of Campylobacter duck isolates from South Korea

Province Chonnam

Chungnam

Chungbuk

Gyonggi

a b

CC

ST

aspA

glnA

gltA

glyA

pgm

tkt

uncA

C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni

ST-21

21 4253 45 4512 3266 5154 5857 7015

4 1 3 1 1 2 1 1

2 2 4 37 8 2 8 350

1 17 7 52 61 1 2 410

1 52 10 16 4 57 80 271

3 3 4 28 64 28 28 82

2 2 1 74 74 58 74 58

1 1 7 29 57 29 24 25

5 5 1 23 23 58 23 23

C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni

ST-21

21 50 4253 45 3784 4536 7016 1034 5231 5096 5917 5857

4 1 1 2 1 1 1 1 1 1 2 2

2 2 2 4 8 37 37 2 8 22 37 8

1 1 17 7 315 52 52 61 61 8 364 2

1 12 52 10 2 4 4 4 4 4 359 80

3 3 3 4 2 64 64 64 28 28 64 28

2 2 2 1 11 129 127 74 74 74 127 74

1 1 1 7 12 29 99 25 25 99 47 24

5 5 5 1 6 23 23 23 23 35 23 23

C. jejuni C. jejuni C. jejuni C. jejuni C. jejuni

ST-21

21 50 45 4852 3784

2 1 1 1 1

2 2 4 37 8

1 1 7 7 315

1 12 10 10 2

3 3 4 4 2

2 2 1 1 11

1 1 7 7 12

5 5 1 1 6

C. jejuni C. jejuni C. jejuni C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli

ST-21 ST-45 U ST-828

21 4852 6149 889 1593 2075 2397 4291 6582 7014 6148

1 1 1 2 1 1 1 1 1 1 1

2 37 37 33 32 124 184 33 33 32 33

1 7 364 39 39 39 39 327 39 39 38

1 10 4 30 30 30 30 30 402 262 30

3 4 28 82 82 79 82 79 82 79 79

2 1 74 113 104 104 113 113 113 104 113

1 7 99 47 43 35 43 43 43 43 47

5 1 23 41 17 17 17 17 17 17 17

C. jejuni C. jejuni C. jejuni C. jejuni

ST-21

21 50 696 1332

1 1 2 1

2 2 2 2

1 1 1 1

1 12 4 29

3 3 28 28

2 2 58 58

1 1 25 25

5 5 58 58

a

ST-45 ST-692 ST-1034 ST-1332 U

ST-45 ST-354 ST-692 ST-1034 U

ST-45 ST-354

U

ST-1332

b

U, no clonal complex assigned yet. New STs are shown in bold.

which Campylobacter is almost never detected in animals of ⬍2 weeks of age (31). The reason for the early Campylobacter colonization in ducks is unclear. While evidence exists for chickens (32), maternal antibody resistance to Campylobacter colonization is unknown for ducks. Other studies have reported that different life cycles and high levels of environmental contamination may explain the early colonization (13, 33), or ducks may be a prime host compared to chickens (34). Antibiotic resistance, particularly multidrug resistance, is a public health problem. In our study, 44 (91.4%) C. jejuni strains and all C. coli strains were multidrug resistant. Ducks may play a role in transmitting multidrug-resistant Campylobacter to hu-

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mans along the food chain, like the case for chickens (8). In addition, one strain of C. jejuni and one C. coli strain were resistant to all eight antimicrobials isolated from ducks. These observations highlight the need for rigorous surveillance of antibiotics used in ducks to control further emergence of antibiotic-resistant Campylobacter. Our results indicate high-level resistance of the microorganisms from ducks to ciprofloxacin and nalidixic acid. The prevalence of fluoroquinolone-resistant Campylobacter varies greatly among different countries. No fluoroquinolone-resistant C. jejuni is found in Tanzania (35), whereas higher resistances (17.4 to 76%) to ciprofloxacin have been reported in other areas (14, 16,

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Chonbuk

MLST allelic profile

No. of isolates

Campylobacter species

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TABLE 4 Distribution of MLST profiles and antimicrobial resistance patterns among C. jejuni and C. coli isolates from ducks Species

CCa

STb

No. of isolates

Antimicrobial resistance pattern

C. jejuni

ST-21 ST-21 ST-21 ST-21 ST-21 ST-21 ST-21 ST-21 ST-21 ST-21 ST-21 ST-21 ST-21 ST-45 ST-45 ST-45 ST-45 ST-45 ST-45 ST-45 ST-354 ST-354 ST-692 ST-692 ST-692 ST-1034 ST-1034 ST-1034 ST-1332 ST-1332 ST-1332 ST-1332 ST-1332 U U U U U U

21 21 21 21 21 21 21 21 50 50 50 4253 4253 45 45 45 45 45 4852 4852 3784 3784 4512 4536 7016 1034 3266 5231 696 696 1332 5154 5154 5096 5857 5857 5917 6149 7015

1 1 2 1 3 1 2 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1

Susceptible Amp Azi Cip Gen Nal Tet Amp Azi Cip Nal Tet Amp Cip Nal Amp Cip Nal Tet Amp Ery Tet Cip Nal Tet Nal Amp Azi Cip Ery Nal Tet Amp Azi Cip Nal Tet Cip Nal Tet Amp Cip Cli Gen Nal Tet Cip Nal Tet Amp Azi Tet Amp Cip Nal Tet Amp Tet Cip Nal Cip Nal Tet Amp Cip Tet Cip Nal Tet Amp Azi Cip Cli Ery Gen Nal Tet Amp Cip Nal Cip Nal Tet Azi Cip Cli Ery Nal Tet Cip Nal Tet Amp Azi Cip Nal Amp Cip Nal Tet Amp Cip Nal Tet Amp Cip Nal Tet Cip Nal Tet Cip Amp Cip Nal Tet Tet Cip Nal Tet Amp Cip Gen Nal Tet Amp Cip Nal Tet Amp Cip Nal Tet Amp Cip Nal Tet Amp Azi Cip Ery Nal Tet

C. coli

ST-828 ST-828 ST-828 ST-828 ST-828 ST-828 ST-828 ST-828 U

889 889 1593 2075 2397 4291 6582 7014 6148

1 1 1 1 1 1 1 1 1

Amp Azi Cip Cli Ery Gen Nal Tet Amp Cip Nal Tet Amp Azi Ery Tet Amp Cip Nal Tet Amp Cip Amp Cip Nal Tet Amp Tet Amp Azi Cip Ery Nal Tet Amp Cip Nal Tet

a b

U, no clonal complex assigned yet. New STs are shown in bold.

served between the STs in our study. All STs showed multidrug resistance, and the majority of the clonal complexes (ST-21, ST45, and ST-828) were multidrug resistant and fluoroquinolone resistant. In particular, the STs that overlapped with humans showed multidrug resistance (Table 4). The high risk to humans is that ST-889 and ST-3784 were resistant to all eight antimicrobials tested. In addition to direct transmission to humans, ducks may

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36). Over 90% C. jejuni resistance to nalidixic acid was reported recently in Vietnam (15). Similarly, C. coli strains with high resistance to fluoroquinolones have been found in Iran, the United Kingdom, Malaysia, and Vietnam, with resistance rates ranging from less than 20% to 100% (11, 14, 15, 36). The high fluoroquinolone resistance rate for Campylobacter in our study may be attributed to the widespread use of fluoroquinolones in poultry production in South Korea before July 2010 (37). Additionally, one study showed that fluoroquinolone-resistant Campylobacter continues to persist even after removal of the selection pressure (38). Moreover, fluoroquinolone-resistant strains enhance the fitness of susceptible strains with no antibiotic selection pressure (39). An unexpected result was that two strains of C. jejuni and one strain of C. coli were resistant to ciprofloxacin but susceptible to nalidixic acid. The same pattern has been reported for C. jejuni and C. coli isolates from swine (40), but the mechanism of resistance to ciprofloxacin and susceptibility to nalidixic acid is unknown. It is well established that point mutations in gyrA can confer resistance to both ciprofloxacin and nalidixic acid or to nalidixic acid alone (41). Other reports show that ciprofloxacin resistance is not inevitable with the gyrA mutation (42). Further investigation is required to fully define the mechanisms involved in ciprofloxacin and nalidixic acid resistance in Campylobacter. MLST is an important tool for elucidating the diversity and transmission routes of Campylobacter isolates for humans. In the present study, ST-21 and ST-45, assigned to CC-21 and CC-45, respectively, were more prevalent than other STs in C. jejuni isolates from South Korean ducks. These two STs are the most common STs in humans (43, 44). Our results are consistent with those of a previous report from the United Kingdom showing that the most common ST from domestic ducks was ST-45 (13). ST-21 from our study was the first to be found largely in ducks compared with previous studies and the MLST database (13, 15, 25). ST-21 has a wide host range in the MLST database, including chickens, cattle, wild birds, and the environment, but only one ST-21 isolate was from ducks. The reasons for the high prevalence (9/46 isolates [19.6%]) of ST-21 isolated from ducks in our study may be that ducks are an ST-21 host and that no large investigations have compared data for ducks with those for other poultry, such as chickens and turkeys, from the MLST database. The increasing fitness of certain STs may have induced the high prevalence in ducks, as poultry may mediate the microevolution of Campylobacter to become widespread (45). Ducks maintained in an open air system can easily exchange pathogens with the environment, wild animals, or other domestic animals near the duck farm, which is consistent with the case for other livestock, such as chickens (46). Ducks are an important reservoir of Campylobacter and may threaten human health (11). In the only report of MLST data from human clinical samples in a restricted area in South Korea, only 2 common STs (ST-21 and ST-45) isolated from ducks overlapped with human isolates (47). However, 53.5% (15/28 STs) of the STs found in ducks were previously associated with human infections. In particular, three STs of C. jejuni (ST-3784, ST-3266, and ST5154) and four STs of C. coli (ST-1593, ST-2075, ST-2397, and ST-4291) were particularly associated with human infections with ducks as the only source. Therefore, duck-associated Campylobacter poses a plausible risk to human health. An infection with an antimicrobial-resistant Campylobacter strain may lead to a suboptimal outcome or treatment failure (48). No significant differences in antimicrobial resistance were ob-

Campylobacter spp. Isolated from Ducks in South Korea

ACKNOWLEDGMENTS This publication made use of the Campylobacter MLST website (http: //pubmlst.org/campylobacter/) developed by Keith Jolley and sited at the University of Oxford (25). The development of this site was funded by the Wellcome Trust. This study was supported by the Cooperative Research Program for Agriculture Science & Technology Development (grant PJ010114), Rural Development Administration, and by the Bio-Industry Technology Development Program (grant 314004-3), Ministry for Agriculture, Food and Rural Affairs, Republic of Korea.

REFERENCES 1. Coker AO, Isokpehi RD, Thomas BN, Amisu KO, Obi CL. 2002. Human campylobacteriosis in developing countries. Emerg. Infect. Dis. 8:237–244. http://dx.doi.org/10.3201/eid0803.010233. 2. Eurosurveillance Editorial Team. 2012. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2010. Euro Surveill. 17:20113. http://www.eurosurveillance .org/ViewArticle.aspx?ArticleId⫽20113. 3. Nachamkin I, Allos BM, Ho T. 1998. Campylobacter species and Guillain-Barre syndrome. Clin. Microbiol. Rev. 11:555–567. 4. Young KT, Davis LM, DiRita VJ. 2007. Campylobacter jejuni: molecular biology and pathogenesis. Nat. Rev. Microbiol. 5:665– 679. http://dx.doi .org/10.1038/nrmicro1718. 5. Engberg J, Aarestrup FM, Taylor DE, Gerner-Smidt P, Nachamkin I. 2001. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerg. Infect. Dis. 7:24 –34. http://dx.doi.org/10.3201/eid0701.010104. 6. Nachamkin I, Ung H, Li M. 2002. Increasing fluoroquinolone resistance in Campylobacter jejuni, Pennsylvania, U.S.A.,1982-2001. Emerg. Infect. Dis. 8:1501–1503. http://dx.doi.org/10.3201/eid0812.020115. 7. Skjot-Rasmussen L, Ethelberg S, Emborg HD, Agerso Y, Larsen LS, Nordentoft S, Olsen SS, Ejlertsen T, Holt H, Nielsen EM, Hammerum AM. 2009. Trends in occurrence of antimicrobial resistance in Campylobacter jejuni isolates from broiler chickens, broiler chicken meat, and human domestically acquired cases and travel associated cases in Denmark. Int. J. Food Microbiol. 131:277–279. http://dx.doi.org/10.1016 /j.ijfoodmicro.2009.03.006. 8. Kittl S, Kuhnert P, Hachler H, Korczak BM. 2011. Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland. J. Appl. Microbiol. 110: 513–520. http://dx.doi.org/10.1111/j.1365-2672.2010.04906.x. 9. Mullner P, Spencer SE, Wilson DJ, Jones G, Noble AD, Midwinter AC, Collins-Emerson JM, Carter P, Hathaway S, French NP. 2009. Assigning the source of human campylobacteriosis in New Zealand: a comparative genetic and epidemiological approach. Infect. Genet. Evol. 9:1311–1319. http://dx.doi.org/10.1016/j.meegid.2009.09.003. 10. Unicomb LE, Fullerton KE, Kirk MD, Stafford RJ. 2009. Outbreaks of campylobacteriosis in Australia, 2001 to 2006. Foodborne Pathog. Dis. 6:1241–1250. http://dx.doi.org/10.1089/fpd.2009.0300. 11. Little CL, Richardson JF, Owen RJ, de Pinna E, Threlfall EJ. 2008. Prevalence, characterisation and antimicrobial resistance of Campylobacter and Salmonella in raw poultrymeat in the UK, 2003-2005. Int. J. Environ. Health Res. 18:403– 414. http://dx.doi.org/10.1080/09603120802100220.

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12. Abid M, Wimalarathna H, Mills J, Saldana L, Pang W, Richardson JF, Maiden MC, McCarthy ND. 2013. Duck liver-associated outbreak of campylobacteriosis among humans, United Kingdom, 2011. Emerg. Infect. Dis. 19:1310 –1313. http://dx.doi.org/10.3201/eid1908.121535. 13. Colles FM, Ali JS, Sheppard SK, McCarthy ND, Maiden MC. 2011. Campylobacter populations in wild and domesticated Mallard ducks (Anas platyrhynchos). Environ. Microbiol. Rep. 3:574 –580. http://dx.doi.org/10 .1111/j.1758-2229.2011.00265.x. 14. Adzitey F, Rusul G, Huda N, Cogan T, Corry J. 2012. Prevalence, antibiotic resistance and RAPD typing of Campylobacter species isolated from ducks, their rearing and processing environments in Penang, Malaysia. Int. J. Food Microbiol. 154:197–205. http://dx.doi.org/10.1016/j .ijfoodmicro.2012.01.006. 15. Carrique-Mas JJ, Bryant JE, Cuong NV, Hoang NV, Campbell J, Hoang NV, Dung TT, Duy DT, Hoa NT, Thompson C, Hien VV, Phat VV, Farrar J, Baker S. 2014. An epidemiological investigation of Campylobacter in pig and poultry farms in the Mekong Delta of Vietnam. Epidemiol. Infect. 142:1425–1436. http://dx.doi.org/10.1017/S0950268813002410. 16. Tsai HJ, Hsiang PH. 2005. The prevalence and antimicrobial susceptibilities of Salmonella and Campylobacter in ducks in Taiwan. J. Vet. Med. Sci. 67:7–12. http://dx.doi.org/10.1292/jvms.67.7. 17. Food and Agriculture Organization (FAO). 2014. FAOSTAT on production of duck meat in the world and Asia. Food and Agriculture Organization, Washington, DC. Accessed 5 February 2014. 18. Food and Agriculture Organization (FAO). 2014. FAOSTAT on production of duck meat in South Korea. Food and Agriculture Organization, Washington, DC. Accessed 5 February 2014. 19. Cha SY, Kang M, Yoon RH, Park CK, Moon OK, Jang HK. 2013. Prevalence and antimicrobial susceptibility of Salmonella isolates in Pekin ducks from South Korea. Comp. Immunol. Microbiol. Infect. Dis. 36: 473– 479. http://dx.doi.org/10.1016/j.cimid.2013.03.004. 20. Linton D, Owen RJ, Stanley J. 1996. Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals. Res. Microbiol. 147:707–718. http://dx.doi.org/10 .1016/S0923-2508(97)85118-2. 21. Wang G, Clark CG, Taylor TM, Pucknell C, Barton C, Price L, Woodward DL, Rodgers FG. 2002. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus. J. Clin. Microbiol. 40:4744 – 4747. http: //dx.doi.org/10.1128/JCM.40.12.4744-4747.2002. 22. National Antimicrobial Resistance Monitoring System (NARMS). 2011. NARMS retail meat annual report, 2011. US Food and Drug Administration, Washington, DC. 23. Clinical and Laboratory Standards Institute (CLSI). 2011. Performance standards for antimicrobial susceptibility testing; 21st informational supplement. CLSI document M100-S21. CLSI, Wayne, PA. 24. Dingle KE, Colles FM, Wareing DR, Ure R, Fox AJ, Bolton FE, Bootsma HJ, Willems RJ, Urwin R, Maiden MC. 2001. Multilocus sequence typing system for Campylobacter jejuni. J. Clin. Microbiol. 39:14 –23. http://dx .doi.org/10.1128/JCM.39.1.14-23.2001. 25. Jolley KA, Maiden MCJ. 2010. BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics 11:595. http://dx.doi.org/10.1186/1471-2105-11-595. 26. Chen X, Naren GW, Wu CM, Wang Y, Dai L, Xia LN, Luo PJ, Zhang Q, Shen JZ. 2010. Prevalence and antimicrobial resistance of Campylobacter isolates in broilers from China. Vet. Microbiol. 144:133–139. http: //dx.doi.org/10.1016/j.vetmic.2009.12.035. 27. Haruna M, Sasaki Y, Murakami M, Ikeda A, Kusukawa M, Tsujiyama Y, Ito K, Asai T, Yamada Y. 2012. Prevalence and antimicrobial susceptibility of Campylobacter in broiler flocks in Japan. Zoonoses Public Health 59:241–245. http://dx.doi.org/10.1111/j.1863-2378.2011.01441.x. 28. French N, Barrigas M, Brown P, Ribiero P, Williams N, Leatherbarrow H, Birtles R, Bolton E, Fearnhead P, Fox A. 2005. Spatial epidemiology and natural population structure of Campylobacter jejuni colonizing a farmland ecosystem. Environ. Microbiol. 7:1116 –1126. http://dx.doi.org /10.1111/j.1462-2920.2005.00782.x. 29. Sasaki Y, Tsujiyama Y, Tanaka H, Yoshida S, Goshima T, Oshima K, Katayama S, Yamada Y. 2011. Risk factors for Campylobacter colonization in broiler flocks in Japan. Zoonoses Public Health 58:350 –356. http: //dx.doi.org/10.1111/j.1863-2378.2010.01370.x. 30. Kasrazadeh M, Genigeorgis C. 1987. Origin and prevalence of Campylobacter jejuni in ducks and duck meat at the farm and processing plant level. J. Food Prot. 50:321–326.

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also act as vehicles of disease in contact with other domestic animals, such as occurs with other human-pathogenic etiologies (49, 50). The rapid and widespread dissemination of multidrug-resistant Campylobacter clonal groups highlights the need to develop effective infection control measures for these groups in animal reservoir populations. In conclusion, our results highlight the high level of contamination by Campylobacter in South Korean duck farms and the high resistance to antimicrobials in the fluoroquinolone family and show that South Korean ducks are a potentially important source of human infection. These results indicate the need to strengthen implementation of specific control procedures to decrease Campylobacter contamination of ducks.

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aem.asm.org

41.

42.

43. 44. 45.

46.

47.

48.

49. 50.

Prev. Vet. Med. 109:152–157. http://dx.doi.org/10.1016/j.prevetmed.2012 .09.010. Jesse TW, Englen MD, Pittenger-Alley LG, Fedorka-Cray PJ. 2006. Two distinct mutations in gyrA lead to ciprofloxacin and nalidixic acid resistance in Campylobacter coli and Campylobacter jejuni isolated from chickens and beef cattle. J. Appl. Microbiol. 100:682– 688. http://dx.doi.org/10 .1111/j.1365-2672.2005.02796.x. Piddock LJ, Ricci V, Pumbwe L, Everett MJ, Griggs DJ. 2003. Fluoroquinolone resistance in Campylobacter species from man and animals: detection of mutations in topoisomerase genes. J. Antimicrob. Chemother. 51:19 –26. http://dx.doi.org/10.1093/jac/dkg033. Colles FM, Maiden MC. 2012. Campylobacter sequence typing databases: applications and future prospects. Microbiology 158:2695–2709. http://dx .doi.org/10.1099/mic.0.062000-0. Griekspoor P, Engvall EO, Olsen B, Waldenstrom J. 2010. Multilocus sequence typing of Campylobacter jejuni from broilers. Vet. Microbiol. 140:180 –185. http://dx.doi.org/10.1016/j.vetmic.2009.07.022. Asakura H, Taguchi M, Ekawa T, Yamamoto S, Igimi S. 2013. Continued widespread dissemination and increased poultry host fitness of Campylobacter jejuni ST-4526 and ST-4253 in Japan. J. Appl. Microbiol. 114: 1529 –1538. http://dx.doi.org/10.1111/jam.12147. de Haan CP, Lampen K, Corander J, Hanninen ML. 2013. Multilocus sequence types of environmental Campylobacter jejuni isolates and their similarities to those of human, poultry and bovine C. jejuni isolates. Zoonoses Public Health 60:125–133. http://dx.doi.org/10.1111/j.1863-2378 .2012.01525.x. Shin E, Oh Y, Kim M, Jung J, Lee Y. 2013. Antimicrobial resistance patterns and corresponding multilocus sequence types of the Campylobacter jejuni isolates from human diarrheal samples. Microb. Drug Resist. 19:110 –116. http://dx.doi.org/10.1089/mdr.2012.0099. Engberg J, Neimann J, Nielsen EM, Aerestrup FM, Fussing V. 2004. Quinolone-resistant Campylobacter infections: risk factors and clinical consequences. Emerg. Infect. Dis. 10:1056 –1063. http://dx.doi.org/10 .3201/eid1006.030669. Guan Y, Webby R, Capua I, Waldenstrom J. 2012. H5N1: how to track a flu virus. Nature 483:535–536. http://dx.doi.org/10.1038/483535a. Liu JH, Wei SY, Ma JY, Zeng ZL, Lu DH, Yang GX, Chen ZL. 2007. Detection and characterisation of CTX-M and CMY-2 beta-lactamases among Escherichia coli isolates from farm animals in Guangdong Province of China. Int. J. Antimicrob. Agents 29:576 –581. http://dx.doi.org/10 .1016/j.ijantimicag.2006.12.015.

Applied and Environmental Microbiology

Downloaded from http://aem.asm.org/ on December 5, 2014 by ST ANDREWS UNIV

31. Gregory E, Barnhart H, Dreesen DW, Stern NJ, Corn JL. 1997. Epidemiological study of Campylobacter spp. in broilers: source, time of colonization, and prevalence. Avian Dis. 41:890 – 898. http://dx.doi.org/10 .2307/1592343. 32. Sahin O, Luo N, Huang S, Zhang Q. 2003. Effect of Campylobacter specific maternal antibodies on Campylobacter jejuni colonization in young chickens. Appl. Environ. Microbiol. 69:5372–5379. http://dx.doi .org/10.1128/AEM.69.9.5372-5379.2003. 33. Cawthraw SA, Newell DG. 2010. Investigation of the presence and protective effects of maternal antibodies against Campylobacter jejuni in chickens. Avian Dis. 54:86 –93. http://dx.doi.org/10.1637/9004-072709 -Reg.1. 34. McCrea BA, Tonooka KH, VanWorth C, Atwill ER, Schrader JS. 2006. Colonizing capability of Campylobacter jejuni genotypes from lowprevalence avian species in broiler chickens. J. Food Prot. 69:417– 420. 35. Nonga HE, Muhairwa AP. 2010. Prevalence and antibiotic susceptibility of thermophilic Campylobacter isolates from free range domestic duck (Cairina moschata) in Morogoro Municipality, Tanzania. Trop. Anim. Health Prod. 42:165–172. http://dx.doi.org/10.1007/s11250-009-9401-0. 36. Rahimi E, Alian F, Alian F. 2011. Prevalence and characteristic of Campylobacter species isolated from raw duck and goose meat in Iran, p 171– 175. In Proceedings of the International Conference on Food Engineering and Biotechnology (ICFEB 2011), Singapore. IACSIT Press, Singapore, Republic of Singapore. 37. Ku BK, Kim HJ, Lee YJ, Kim YI, Choi JS, Park MY, Kwon JW, Nam HM, Kim YH, Jung SC, Lee SJ, Kim SH, Kim JH. 2011. Genetic characterization and antimicrobial susceptibility of Campylobacter spp. isolated from domestic and imported chicken meats and humans in Korea. Foodborne Pathog. Dis. 8:381–386. http://dx.doi.org/10.1089/fpd .2010.0680. 38. Luangtongkum T, Jeon B, Han J, Plummer P, Logue CM, Zhang Q. 2009. Antibiotic resistance in Campylobacter: emergence, transmission and persistence. Future Microbiol. 4:189 –200. http://dx.doi.org/10.2217 /17460913.4.2.189. 39. Luo N, Pereira S, Sahin O, Lin J, Huang S, Michel L, Zhang Q. 2005. Enhanced in vivo fitness of fluoroquinolone-resistant Campylobacter jejuni in the absence of antibiotic selection pressure. Proc. Natl. Acad. Sci. U. S. A. 102:541–546. http://dx.doi.org/10.1073/pnas.0408966102. 40. von Altrock A, Hamedy A, Merle R, Waldmann KH. 2013. Campylobacter spp.—prevalence on pig livers and antimicrobial susceptibility.

Antimicrobial susceptibility profiles and molecular typing of Campylobacter jejuni and Campylobacter coli isolates from ducks in South Korea.

Campylobacter is a food-borne zoonotic pathogen that causes human gastroenteritis worldwide. Campylobacter bacteria are commensal in the intestines of...
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