High Prevalence of Fluoroquinolone-Resistant CampylobacterBacteria in Sheep and Increased Campylobacter Counts in theBile and Gallbladders of Sheep Medicated with Tetracycline inFeed

Jing Xia,a,b Jinji Pang,a Yizhi Tang,a* Zuowei Wu,a Lei Dai,a Kritika Singh,a Changyun Xu,a Brandon Ruddell,a

Amanda Kreuder,c Lining Xia,a* Xiaoping Ma,a* Kelly S. Brooks,c Melda M. Ocal,a Orhan Sahin,c Paul J. Plummer,a,c

Ronald W. Griffith,a Qijing Zhanga

aDepartment of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USAbNational Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University,Guangzhou, China

cDepartment of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA

ABSTRACT Campylobacter is a major foodborne pathogen in humans and a signifi-cant cause of abortion in sheep. Although ruminants are increasingly recognized asimportant reservoirs for Campylobacter species, limited information is available aboutthe molecular epidemiology and antimicrobial resistance (AMR) profiles of sheepCampylobacter. Here, we describe a two-trial study that examined Campylobacterprofiles in sheep and determined whether in-feed tetracycline (TET) influenced thedistribution and AMR profiles of Campylobacter. Each trial involved 80 commercialsheep naturally infected with Campylobacter: 40 of these sheep were medicated withtetracycline in feed, while the other 40 received feed without antibiotics. Fecal andbile samples were collected for the isolation of Campylobacter. The bacterial isolateswere analyzed for antimicrobial susceptibility and genotypes. The results revealedthat 87.0% and 61.3% of the fecal and bile samples were positive for Campylobacter(Campylobacter jejuni and Campylobacter coli), with no significant differences be-tween the medicated and nonmedicated groups. All but one of the tested Campylo-bacter isolates were resistant to tetracycline. Although fluoroquinolone (FQ) resis-tance remained low in C. jejuni (1.7%), 95.0% of the C. coli isolates were resistant toFQ. Genotyping revealed that C. jejuni sequence type 2862 (ST2862) and C. coliST902 were the predominant genotypes in the sheep. Feed medication with tetracy-cline did not affect the overall prevalence, species distribution, and AMR profiles ofCampylobacter, but it did increase the total Campylobacter counts in bile and gall-bladder. These findings identify predominant Campylobacter clones, reveal the highprevalence of FQ-resistant C. coli, and provide new insights into the epidemiology ofCampylobacter in sheep.

IMPORTANCE Campylobacter is a major cause of foodborne illness in humans, andantibiotic-resistant Campylobacter is considered a serious threat to public health inthe United States and worldwide. As a foodborne pathogen, Campylobacter com-monly exists in the intestinal tract of ruminant animals, such as sheep and cattle. Re-sults from this study reveal the predominant genotypes and high prevalence of tet-racycline (TET) and fluoroquinolone (FQ) resistance in sheep Campylobacter. Thefinding on fluoroquinolone resistance in sheep Campylobacter is unexpected, as thisclass of antibiotics is not used for sheep in the United States, and it may suggestthe transmission of fluoroquinolone-resistant Campylobacter from cattle to sheep.Additionally, the results demonstrate that in-feed medication with tetracycline in-creases Campylobacter counts in gallbladders, suggesting that the antibiotic pro-

Citation Xia J, Pang J, Tang Y, Wu Z, Dai L,Singh K, Xu C, Ruddell B, Kreuder A, Xia L, Ma X,Brooks KS, Ocal MM, Sahin O, Plummer PJ,Griffith RW, Zhang Q. 2019. High prevalence offluoroquinolone-resistant Campylobacterbacteria in sheep and increased Campylobactercounts in the bile and gallbladders of sheepmedicated with tetracycline in feed. ApplEnviron Microbiol 85:e00008-19. https://doi.org/10.1128/AEM.00008-19.

Editor Donald W. Schaffner, Rutgers, The StateUniversity of New Jersey

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Qijing Zhang,zhang123@iastate.edu.

* Present address: Yizhi Tang, Animal DiseasePrevention and Food Safety, Key Laboratory ofSichuan Province, College of Life Science,Sichuan University, Chengdu, China; Lining Xia,College of Veterinary Medicine, XinjiangAgricultural University, Urumqi, China; XiaopingMa, Key Laboratory of Animal Disease andHuman Health of Sichuan Province, College ofVeterinary Medicine, Sichuan AgriculturalUniversity, Chengdu, China.

J.X. and J.P. contributed equally to this article.

Received 2 January 2019Accepted 22 March 2019

Accepted manuscript posted online 29March 2019Published

FOOD MICROBIOLOGY

crossm

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 1Applied and Environmental Microbiology

16 May 2019

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

motes Campylobacter colonization of the gallbladder. These findings provide new in-formation on Campylobacter epidemiology in sheep, which may be useful forcurbing the spread of antibiotic-resistant Campylobacter in animal reservoirs.

KEYWORDS Campylobacter, antimicrobial resistance, fluoroquinolone, genotype,sheep

Campylobacter species, particularly Campylobacter jejuni and Campylobacter coli, area leading cause of bacterial foodborne gastroenteritis in humans around the world

(1–3). Among all the causes of laboratory-confirmed bacterial foodborne illnesses,Campylobacter was the leading cause (19.2 per 100,000 population) in the year 2017,based on a report by the Centers for Disease Control and Prevention (CDC) FoodNetsurveillance program in the United States (4). Although most Campylobacter-relatedillnesses in humans are characterized as self-limiting diarrhea (watery and/or bloody),antibiotic treatment is utilized in more severe clinical conditions, especially in young,elderly, or immunocompromised patients (5). However, antimicrobial resistance inCampylobacter is increasingly prevalent and has become a major public health concernin both developed and developing counties (6, 7). Of particular concern is the risingresistance to fluoroquinolone (FQ) and macrolide antibiotics, which are clinically im-portant for the treatment of Campylobacter-induced diarrhea in humans, although insome cases tetracyclines (TET) and gentamicin (GEN) are also used to treat systemicinfection caused by Campylobacter (6, 8, 9).

Food-producing animals are important reservoirs for Campylobacter (10). Epidemi-ologically, poultry meat is considered the major source of infection for human campy-lobacteriosis (11). C. jejuni and C. coli frequently colonize the intestines of many speciesof poultry, especially commercial chickens and turkeys, usually as part of the normalmicrobial flora without causing clinical disease (12). Recently, the role of ruminants(cattle and sheep) in Campylobacter ecology has been increasingly recognized indifferent countries (13–17). Ruminant Campylobacter strains can be transmitted tohumans via contaminated milk and water, environmental contamination, or directcontact with animals (18). Source attribution studies indicate that ruminant Campylo-bacter is a significant source of infection for human campylobacteriosis (19–21).

Typically, Campylobacter colonizes the intestinal tract in animals, but it may alsotranslocate across the intestinal epithelial barrier and causes systemic infection, leadingto bacteremia and abortion in ruminants and even occasionally in humans (1, 22). Infact, Campylobacter is the major cause of ovine abortions worldwide, including in theUnited States. Notably, a single hypervirulent tetracycline-resistant C. jejuni clone(named clone SA, for sheep abortion) is responsible for the majority of Campylobacter-associated ovine abortions in the United States (17, 23, 24). A previous survey of healthysheep in a slaughterhouse revealed genetically diverse C. jejuni strains (including cloneSA) present in the intestinal tract and gallbladder (25). Despite the fact that ruminantsare increasingly recognized as important reservoirs for Campylobacter, limited informa-tion is available about the molecular epidemiology and antimicrobial resistance profilesof sheep Campylobacter in the United States.

For the control of abortion and other diseases, the tetracycline class of antibiotics iscommonly used in the United States, and it is the only class of antibiotics approved forprevention and control of sheep abortion associated with Campylobacter (23). However,whether the administration of tetracycline in feed impacts the prevalence of Campy-lobacter in sheep is not known. Previously, it was shown that sheep harbored diversestrains of C. jejuni that were commonly resistant to tetracycline; however, resistance toFQ was seldomly detected (25). Recently, FQ resistance in cattle Campylobacter isolateshas increased substantially in the United States (15, 26–28). However, detection of FQresistance in sheep Campylobacter isolates is still limited. To address these knowledgegaps, we collected samples and isolated Campylobacter from controlled treatment (feedmedication with tetracycline) studies using sheep that were derived from commercialoperations and were naturally infected by Campylobacter. The goals were to examine

Xia et al. Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 2

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

the extent of FQ resistance in sheep Campylobacter and determine the effect of in-feedtetracycline on the prevalence, species distribution, and antimicrobial resistance pro-files of Campylobacter in sheep.

RESULTSPrevalence of Campylobacter in sheep. In total, 461 sheep fecal samples and 160

bile samples were collected for isolation of Campylobacter in the two separate trials. Theoverall prevalence rate of Campylobacter in sheep feces was 87.0% (401/461). Amongthe positive samples, 68.6% (275/401) and 37.4% (150/401) were positive for C. jejuniand C. coli, respectively, and 24 fecal samples were positive for both C. jejuni and C. coli(Table 1). The overall prevalence of Campylobacter in fecal samples was comparable(71.8% versus 65.3% for C. jejuni; 33.2% versus 41.7% for C. coli) between the feed-medicated and nonmedicated groups (P � 0.05) (Table 1). Among the bile samples,61.3% (98/160) were positive for Campylobacter (Table 2). Of the Campylobacter isolates,98.0% (96/98) were C. jejuni, and 2.0% (2/98) were C. coli. The two C. coli isolates frombile were both isolated from the feed-medicated group in trial 2 (Table 2). Again, therewas no significant difference (P � 0.05) between the groups in the prevalence of eitherC. jejuni (100% versus 95.8%) or C. coli (0% versus 4.2%). These results indicate that thein-feed tetracycline medication did not affect the overall prevalence of Campylobacterin fecal and bile samples.

Statistically, the overall Campylobacter prevalence was higher in fecal samples thanin bile samples (87.0% versus 61.3%, P � 0.05). However, the proportion of C. jejuni wassignificantly (P � 0.05) lower in feces than in bile (68.6% versus 98.0%), while theproportion of C. coli was higher in feces than in bile (37.4% versus 2.0%). Detailedinformation about the prevalence of Campylobacter and the distribution of C. jejuni andC. coli is summarized in Tables 1 and 2. We analyzed all of the C. jejuni isolates fromfeces and bile by PCR for identification of C. jejuni clone SA, and none were positive,indicating that clone SA was not present in the sheep used in this study.

Quantitative measurement of Campylobacter CFU in bile and gallbladder mu-cosa. Significant differences in Campylobacter numbers (CFU/ml) between the feed-medicated and nonmedicated groups were detected for both the bile and gallbladdermucosal samples, with numbers higher in the medicated group (Fig. 1). In the bilesamples, the mean log CFU/ml � standard error of the mean (SEM) values were5.041 � 0.281 and 6.021 � 0.108 in the nonmedicated and medicated groups, respec-tively, indicating an almost 10-fold difference. The difference between the twogroups was statistically significant [t(3.252,6) � 0.0174, P � 0.05]. For the gallbladdermucosal samples, the mean log CFU/ml � SEM of gallbladder mucosal sampleswere 4.7239 � 0.2932 and 5.823 � 0.2611 in the nonmedicated and medicatedgroups, respectively, again indicating an approximately 10-fold difference. Thedifference was also statistically significant [t(2.722,57) � 0.0086, P � 0.05]. Theseresults indicated that Campylobacter titers are higher in the gallbladder and bilesamples taken from the feed medicated group.

Antimicrobial susceptibility of the Campylobacter isolates. For the tested fecal C.jejuni isolates (n � 236), all (100%) were resistant to tetracycline, but the rates ofresistance to ciprofloxacin (CIP) and nalidixic acid (NAL) were low, at 1.7% (4/236) and3.0% (7/236), respectively. All four ciprofloxacin-resistant (CIPr) C. jejuni isolates werederived from trial 2. One C. jejuni isolate from the nonmedicated group in trial 1 showedresistance to gentamicin. The fecal C. coli isolates also showed a high resistance rate totetracycline (99.0%; 100/101), with only 1 isolate sensitive to this antibiotic. In contrastto C. jejuni, the C. coli isolates were highly resistant to ciprofloxacin (95.0%; 96/101) andnalidixic acid (95.0%; 96/101). Resistance to the other five antibiotics was not observed.The key MIC results are shown in Table 3, while other relevant MIC data (MIC ranges,MIC50, and MIC90) are presented in Table S1.

Among the isolates from bile samples, all tested C. jejuni isolates were resistant totetracycline (100.0%; 96/96), but susceptible to other antibiotics, while the two C. coliisolates were resistant to tetracycline, ciprofloxacin, and nalidixic acid. None of the bile

Campylobacter in Sheep Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 3

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

TAB

LE1

Isol

atio

nra

tes

ofCa

mpy

loba

cter

insh

eep

feca

lsa

mp

les

Isol

ate

gro

up

No.

(%)

ofis

olat

esin

tria

la:

1(2

1Ju

ne

2017

–6Ju

ly20

17)

2(1

3Ju

ly20

17–2

6Ju

ly20

17)

All

feca

lsa

mp

les

1st

wee

k(2

1Ju

ne)

2nd

wee

k(2

9Ju

ne)

3rd

wee

k(6

July

)1s

tw

eek

(13

July

)2n

dw

eek

(21

July

)3r

dw

eek

(26

July

)

NM

MT

NM

MT

NM

MT

NM

MT

NM

MT

NM

MT

NM

MTb

All

isol

ates

34(5

2.3)

31(4

7.7)

65(1

00)

34(5

0.0)

34(5

0.0)

68(1

00)

32(5

1.6)

30(4

8.4)

62(1

00)

36(5

1.4)

34(4

8.6)

70(1

00)

33(5

0.0)

33(5

0.0)

66(1

00)

33(4

7.1)

37(5

2.9)

70(1

00)

202

(50.

4)19

9(4

9.6)

401

(100

)C.

jeju

ni30

(58.

8)21

(41.

2)51

(78.

5)27

(49.

1)28

(50.

9)55

(80.

9)20

(46.

5)23

(53.

5)43

(69.

4)19

(51.

4)18

(48.

6)37

(52.

9)24

(55.

8)19

(44.

2)43

(65.

2)25

(54.

3)21

(45.

7)46

(65.

7)14

5(5

2.7)

130

(47.

3)27

5(6

8.6)

C.co

li5

(29.

4)12

(70.

6)17

(26.

2)8

(47.

1)9

(52.

9)17

(25.

0)13

(56.

5)10

(43.

5)23

(37.

1)19

(52.

8)17

(47.

2)36

(51.

4)10

(40.

0)15

(60.

0)25

(37.

9)12

(37.

5)20

(62.

5)32

(45.

7)67

(44.

7)83

(55.

3)15

0(3

7.4)

aN

M,n

onm

edic

ated

grou

p;M

,med

icat

edgr

oup

;T,t

otal

.b24

feca

lsa

mp

les

wer

ep

ositi

veb

oth

for

C.je

juni

and

C.co

li.

Xia et al. Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 4

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

C. jejuni or C. coli isolates showed resistance to gentamicin, azithromycin (AZI), eryth-romycin (ERY), florfenicol (FFN), telithromycin (TEL), or clindamycin (CLI).

Notably, for the fecal samples, the ciprofloxacin (CIP) resistance rate (95.0%) in C. coliis significantly (P � 0.05) higher than that in C. jejuni (1.7%). There is no significantdifference (P � 0.05) in the overall antimicrobial susceptibility profiles, as tested in thisstudy, between fecal and bile C. jejuni. The two C. coli isolates from bile samples alsoshowed similar resistance patterns to the C. coli isolates from feces. In addition, therewas also no significant difference (P � 0.05) in the antimicrobial resistance rates of C.jejuni and C. coli between the nonmedicated and feed-medicated groups.

Genetic diversity of the Campylobacter isolates from sheep. In total, 24 C. coliand 39 C. jejuni isolates were selected for pulsed-field gel electrophoresis (PFGE)analysis. The 24 C. coli isolates consisted of twelve CIPr C. coli isolates from feces (onerepresentative isolate for each group and each sampling time in the two trials), the twoCIPr C. coli isolates from bile, the five ciprofloxacin-susceptible (CIPs) fecal C. coli isolates,the TET-susceptible (TETs) C. coli isolate, and the other four fecal C. coli isolates (isolatedfrom the same sheep with the two CIPr C. coli isolates from bile). The 24 C. coli isolateswere grouped into three separate clusters, with the vast majority of isolates (75.0%;18/24) grouped into type III (Fig. 2). The five CIPs C. coli isolates were all type I, and thetype II group included one CIPr C. coli isolate from feces. The predominant cluster (typeIII) consisted of 18 CIPr C. coli isolates, among which 16 were from fecal samples(including one TET-susceptible isolate and 4 from the same sheep from which the twoCIPr bile isolates were obtained) and two were from bile samples (Fig. 2). Multilocussequence typing (MLST) analysis showed that the C. coli isolates grouped in types I, II,and III belong to ST827, ST1068, and ST902, respectively (Fig. 2), consistent with theclassifications using PFGE. Thus, ST902 was the most prevalent ST in the sheep C. coliisolates analyzed in this study. Interestingly, the CIPr bile and fecal isolates from thesame sheep shared the same PFGE type (III) and MLST type (ST902), indicating that thebile and fecal isolates are genetically related.

TABLE 2 Overall prevalence of Campylobacter in sheep bile samplesa

Isolate group

No. (%) of isolates in trialb:

1 (21 June 2017–6 July 2017) 2 (13 July 2017–26 July 2017) All bile samples

NM M T NM M T NM M T

Total 28 (50.9) 27 (49.1) 55 (100) 22 (51.2) 21 (48.8) 43 (100) 50 (51.0) 48 (49.0) 98 (100)C. jejuni 28 (50.9) 27 (49.1) 55 (100) 22 (53.7) 19 (46.3) 41 (95.3) 50 (52.1) 46 (47.9) 96 (98.0)C. coli 0 (0) 0 (0) 0 (0) 0 (0) 2 (100.0) 2 (4.7) 0 (0.0) 2 (100.0) 2 (2.0)aBile samples were collected at necropsy at the end of the two trials.bNM, nonmedicated group; M, medicated group; T, total.

FIG 1 Campylobacter counts (log CFU/ml) in bile (a) and gallbladder mucosal (b) samples in thenonmedicated and medicated groups. Each bar represents the average log CFU per ml (� standard errorof the mean [SEM]). An asterisk (*) indicates a significant difference from the nonmedicated group(P � 0.05). The data include samples from both trials.

Campylobacter in Sheep Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 5

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

The 39 C. jejuni isolates included 12 CIPs C. jejuni isolates from bile (three represen-tative isolates for each group in two trials), the four CIPr C. jejuni isolates from feces, thethree CIPs fecal C. jejuni isolates (isolated from the same sheep with the CIPr isolates),20 CIPs fecal C. jejuni isolates (from different sheep from which CIPr isolates wereisolated, representative isolates from each group and each sampling time in two trials,and including ten selected fecal C. jejuni isolates from the same sheep with the six bileC. jejuni isolates). The 39 C. jejuni isolates were clustered into five different clusters (Fig.3), apart from four isolates that were nontypeable by PFGE because they were notdigestible by KpnI. The four nontypeable isolates included one CIPs bile isolate andthree selected CIPs fecal isolates from the same three sheep with bile isolates. Type Awas the predominant genotype and accounted for 69.2% (27/39) of the analyzedisolates. This predominant cluster included both CIPr and CIPs C. jejuni isolates fromfecal and bile samples. MLST results indicated that all but two type A C. jejuni isolatesbelonged to ST2862 (Fig. 2). One of the two non-ST2862 isolates was a CIPr C. jejuniisolate (identifier [ID] 0993), which showed only one base difference in gltA from thatof ST2862. After the sequence was submitted to the MLST database, the new gltA allelewas assigned to gltA586 (in contrast to gltA2 in ST2862), and the isolate was assigneda new ST, ST9380. The other non-ST2862 isolate was also a new ST (ST9379) and wasa bile isolate (ID 292). Detailed information for MLST profiles of the two isolates ispresented in Table S2. The other four PFGE patterns, types B, C, D, and E, included 8 C.jejuni isolates. Type B belonged to ST982, while types C, D, and E all belonged to ST52(Fig. 3). For the selected bile and fecal C. jejuni isolates from the same sheep, thesituation was more diverse than that with the C. coli isolates (Fig. 3). Bile and fecalisolates from each of the animals with ID numbers 898, 346, and 604 shared the samePFGE and MLST profiles, while the bile and fecal isolates from three other animals wereof different PFGE or MLST types (ID 292, the same PFGE with different STs; ID 961,different PFGE types but with the same ST; ID 610, different PFGE types with differentSTs).

Together, the PFGE and MLST findings indicated predominant genotypes in both C.coli and C. jejuni, suggesting that clonal expansion may have been involved in thedissemination of both C. coli and C. jejuni in sheep.

Point mutations in gyrA. Based on different PFGE and MLST types, eight CIPr C. coliand four CIPr C. jejuni isolates were selected for determination of the point mutationsin gyrA. All of the CIPr C. coli isolates harbored a single Thr-86-Ile mutation in GyrA

TABLE 3 Antimicrobial resistance profiles and rates of the tested Campylobacter isolates from fecal samples

Trial no. Species Medication group

Isolates (% [no./total tested]) resistant to:

Tetracycline Ciprofloxacin Nalidixic acid Gentamicin

1 C. jejuni Nonmedicated 100.0 (60/60) 0.0 (0/60) 3.3 (2/60) 1.7 (1/60)Medicated 100.0 (60/60) 0.0 (0/60) 5.0 (3/60) 0.0 (0/60)Total 100.0 (120/120) 0.0 (0/120) 4.2 (5/120) 0.8 (1/120)

C. coli Nonmedicated 100.0 (25/25) 100.0 (25/25) 100.0 (25/25) 0.0 (0/25)Medicated 100.0 (25/25) 100.0 (25/25) 100.0 (25/25) 0.0 (0/25)Total 100.0 (50/50) 100.0 (50/50) 100.0 (50/50) 0.0 (0/50)

2 C. jejuni Nonmedicated 100.0 (58/58) 1.7 (1/58) 3.4 (2/58) 0.0 (0/58)Medicated 100.0 (58/58) 5.2 (3/58) 0.0 (0/58) 0.0 (0/58)Total 100.0 (116/116) 3.4 (4/116) 1.7 (2/116) 0.0 (0/116)

C. coli Nonmedicated 96.0 (24/25) 88.0 (22/25) 88.0 (22/25) 0.0 (0/25)Medicated 100.0 (26/26) 92.3 (24/26) 92.3 (24/26) 0.0 (0/26)Total 98.0 (50/51) 90.2 (46/51) 90.2 (46/51) 0.0 (0/51)

Both trials C. jejuni Nonmedicated 100.0 (118/118) 0.8 (1/118) 3.4 (4/118) 0.8 (1/118)Medicated 100.0 (118/118) 2.5 (3/118) 2.5 (3/118) 0.0 (0/118)Total 100.0 (236/236) 1.7 (4/236) 3.0 (7/236) 0.4 (1/236)

C. coli Nonmedicated 98.0 (49/50) 94.0 (47/50) 94.0 (47/50) 0.0 (0/50)Medicated 100.0 (51/51) 96.1 (49/51) 96.1 (49/51) 0.0 (0/51)Total 99.0 (100/101) 95.0 (96/101) 95.0 (96/101) 0.0 (0/101)

Xia et al. Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 6

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

without any other amino acid changes in this region (Fig. 2). For the CIPr C. jejuniisolates, three had the Thr-86-Ile point mutation alone, and one carried the Thr-86-Ilemutation plus the Ser-22-Gly, Asn-203-Ser, and Arg-285-Lys changes (Fig. 3). Theseresults are consistent with the known role of the Thr-86-Ile mutation in mediating CIPresistance in Campylobacter.

DISCUSSION

Results from this study revealed high prevalences of TET-resistant Campylobacterspp. and CIPr C. coli strains in sheep derived from two commercial farms. These animalswere naturally infected and already carried antibiotic-resistant Campylobacter on theday of arrival (Tables 1, 2, and 3). Subsequent treatment with in-feed tetracycline in aresearch setting did not affect the overall prevalence, antimicrobial susceptibilityprofiles, and species distribution of Campylobacter, as there were no statistically sig-nificant differences (P � 0.05) between the feed-medicated and nonmedicated groups.However, the quantitative CFU counts in bile and gallbladder mucosa collected at theend of the trials revealed higher total CFU counts in the medicated group than in thenonmedicated group (P � 0.05), suggesting that tetracycline treatment may promoteCampylobacter colonization in the gallbladder. The exact reason for this effect isunknown, but it is known that tetracyclines undergo significant enterohepatic circula-tion, resulting in significant concentration of tetracyclines in bile, which may inhibitcompetitive bacterial organisms and confer a colonization advantage for tetracycline-resistant Campylobacter. Since similar numbers of animals received therapeutic treat-

FIG 2 PFGE patterns of representative C. coli isolates in sheep. T1 and T2, trials 1 and 2, respectively; W1, W2, and W3, 1st, 2nd, and 3rd weeks, respectively;CIP, ciprofloxacin; NC, at necropsy; ND, not done. #, TET-susceptible C. coli. Boldface animal identifiers (IDs) indicate animals from which both bile and fecalisolates were typed.

Campylobacter in Sheep Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 7

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

ment in both groups, it is likely that the effect is attributable to in-feed use oftetracycline. Regardless of the reasons, the enhanced colonization of the gallbladdermay give Campylobacter an advantage in the spread to other organs or disseminationback to the intestinal tract.

In fecal samples, C. jejuni accounted for 68.6% of the total isolates (Table 1), whilein bile samples, the isolates were almost exclusively C. jejuni (98.0%), suggesting that C.jejuni has a higher tropism to bile or a higher ability to reach the gallbladder and adaptwithin it. Previous studies observed similar prevalence of C. jejuni and C. coli in sheepgallbladders (29, 30); however, these previous studies were conducted in a differentcountry, and it is possible that differences in sheep production practices or geneticvariability of Campylobacter strains may influence gallbladder colonization by theorganism. Bile is harmful to bacteria; however, Campylobacter utilizes the multidrugefflux pump CmeABC for bile resistance (31–34). The exact reason for the predomi-nance of C. jejuni in the gallbladder, as observed in this study, is unknown and remainsto be investigated in future studies. Interestingly, C. jejuni clone SA was not detected inthe sheep examined in this study. This clone is a predominant cause of Campylobacter-associated ovine abortions and is also distributed in feedlot and dairy cattle in theUnited States (23, 35). A recent study also reported identification of clone SA in Grenada(36). The lack of clone SA detection in this study is not entirely surprising, as we havepreviously found that the distribution of clone SA is highly variable from farm to farm(25). Thus, it was likely that the two farms from which the sheep isolates were deriveddid not harbor this particular C. jejuni strain.

Except for FQs and tetracycline, the Campylobacter isolates examined in this studywere generally susceptible to other tested antimicrobials (Table 3). All but one of thetested Campylobacter isolates from fecal and bile samples were resistant to tetracycline.

FIG 3 PFGE patterns of representative C. jejuni isolates in sheep. T1 and T2, trials 1 and 2, respectively; W1, W2, and W3, 1st, 2nd, and 3rd weeks, respectively;CIP, ciprofloxacin; NC, at necropsy; ND, not done. An asterisk (*) indicates newly assigned STs. Boldface animal IDs indicate animals from which both bile andfecal isolates were typed. Four of the C. jejuni isolates were nontypeable by PFGE and are not shown.

Xia et al. Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 8

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

This finding further strengthens the result of our previous study on Campylobactercarriage in sheep slaughterhouses, which revealed that the rate of resistance totetracycline was already 83.3% (25). In this study, the in-feed tetracycline medicationdid not further influence the prevalence of tetracycline-resistant Campylobacter due tothe fact that tetracycline resistance in the sheep was already very high (virtually 100%)at the beginning of the treatment. Thus, the result should be interpreted cautiously.Given the high prevalence of tetracycline-resistant Campylobacter on sheep farms inthe United States, the use of tetracycline as a means to control Campylobacter-inducedsheep abortion would have a limited impact. In fact, tetracycline medication may havefacilitated the persistence of tetracycline-resistant Campylobacter strains on sheepfarms. Results from this study further underline the need for improved tetracyclinestewardship in sheep to reduce the prevalence of tetracycline-resistant Campylobacter.

A significant finding of this work is the high detected prevalence of CIPr C. coli (95%),whereas CIP resistance in the sheep C. jejuni isolates remained low (1.7%) (Table 3). Thedifference between C. coli and C. jejuni CIP resistance rates is unlikely due to variationin their susceptibility to fluoroquinolones, as CIPr C. jejuni is also common in otheranimal species (6, 7). The high-level prevalence of CIPr C. coli in sheep was unexpected,as this class of antibiotics is not used in sheep production in the United States. A recentstudy on cattle Campylobacter reported CIP resistance rates of 35.4% in C. jejuni and77.3% in C. coli (15). This could be explained by the fact that FQ antibiotics are labeledfor treatment of respiratory disease in cattle in the United States. The reason for thedetected high incidence of CIPr C. coli in sheep in this study is unknown, but there isa possibility of transmission of CIPr Campylobacter from cattle to sheep. Although thesampled sheep farms were not adjacent to other animal species, sheep could getCampylobacter through birds, flies, or other transmission vehicles. However, if this werethe sole case, we would have also observed an increased prevalence of CIPr C. jejuni insheep, as it is also common in cattle. It is possible that the CIPr C. coli strains wereintroduced to sheep by chance and something on these farms favored the selection ofCIPr C. coli, contributing to its overall high prevalence in sheep. It should be pointed outthat fluoroquinolones are also approved for use in swine and were used in poultry priorto 2005 in the United States. Thus, the possibility that sheep acquired CIPr C. coli fromother animal species rather than from cattle cannot be totally excluded.

To determine the genetic relatedness of the Campylobacter isolates, we conductedPFGE and MLST analyses, which are commonly used for genotyping of Campylobacter(20, 37–39). PFGE typing of 24 representative C. coli isolates revealed a limited numberof types and demonstrated that the majority (18/24) of the CIPr C. coli isolates weregrouped into a single type (type III; Fig. 2), which was confirmed by MLST to be a singleST (ST902). It is worthwhile to note that type III included isolates from different andgeographically distant farms and from different sampling times, suggesting that thisgenotype is stable and is prevalent on different sheep farms. Also, bile and fecal isolatesfrom the same sheep shared the same PFGE type and ST. The other two PFGE typesbelonged to ST827 (type I) and ST1068 (type II). C. coli ST827 has been previouslyreported as sometimes the most common ST in both sheep and cattle (40, 41), whileST1068 is more often found in cattle (15, 41, 42). Moreover, ST1068 was also identifiedamong swine C. coli isolates, sometimes as the most predominant ST (42–44). Inaddition, the three identified STs (ST902, ST827, and ST1068) all belong to the ST828complex (Fig. 2). Although the specific prevalent STs vary in different species, C. coliisolates of the ST828 complex, the most prevalent CC (clonal complex) for C. coli, havebeen frequently reported in published studies from different countries and differentsources, including poultry, cattle, sheep, swine, and even humans (15, 45–48). Ourresults confirmed the prevalence of this CC and the existence of specific endemic STsamong animals in different regions.

The 39 representative C. jejuni isolates examined by PFGE typing in this study weregrouped into five clusters (Fig. 3), with the exception of four isolates that were nottypeable by PFGE because KpnI failed to digest appropriately. Similar to the C. coliresult, the majority (27/39) of the tested C. jejuni isolates grouped into one cluster (type

Campylobacter in Sheep Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 9

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

A) and were confirmed by MLST to be a single ST (ST2862) (except for two isolates),suggesting it was the predominant C. jejuni clone in sheep. This clone included bothCIPs and CIPr C. jejuni isolates from both fecal and bile samples that were collected fromdifferent farms (trials 1 and 2) and at different sampling times (Fig. 3), indicating thisgenotype was commonly disseminated in sheep. The other four PFGE types belongedto ST982 (type B) and ST52 (types C, D, and E). ST982 accounted for 20.8% (10/48) ofthe C. jejuni isolates in a previous study involving a lamb slaughterhouse, and it wasalso reported in cattle (15, 25, 49). ST52 was reported sporadically in C. jejuni isolatesfrom different countries and sources, including from ruminants (36–38, 48, 50, 51). BothST2862 and ST982 belong to CC21, which is the most prevalent CC in C. jejuni.

To examine the possible transmission of Campylobacter between sheep and cattle,we selected some cattle Campylobacter isolates identified in our previous study (15) tocompare with the sheep Campylobacter isolates identified in this study. The PFGE andMLST results revealed that ST902 (the predominant C. coli genotype identified in thisstudy in sheep) with the same PFGE pattern was also found in the cattle C. coli isolates(Fig. S1a), although the predominant ST in cattle was ST1068 (15). Here, we alsoidentified ST1068 in sheep C. coli (Fig. 2), which had a PFGE pattern similar to that ofcattle C. coli (Fig. S1b). These findings further suggest the possible transmission offluoroquinolone-resistant (FQr) C. coli (e.g., ST902 and ST1068) between cattle andsheep. In contrast to the C. coli isolates, the predominant C. jejuni genotype in sheep,ST2862 (Fig. 3), was not previously identified in cattle in the United States (15, 26, 49).Although C. jejuni ST982 has been found in cattle (15), PFGE patterns of cattle isolateswere quite different from those of the sheep isolates (Fig. S1c). Thus, the geneticrelatedness between sheep and cattle C. jejuni isolates was not as clear as that for C.coli.

In Campylobacter, FQ resistance is mediated by point mutations in the quinoloneresistance-determining region (QRDR) of DNA gyrase (GyrA) and by the function of theCmeABC multidrug efflux pump (8, 52). The most frequent mutation associated with FQresistance in Campylobacter is Thr-86-Ile, followed by Asp-90-Asn, Thr-86-Lys, Thr-86-Ala, Thr-86-Val, Asp-90-Tyr, and Ala-70-Thr (8, 53). Additionally, double mutations,including Thr-86-Ile plus Pro-104-Ser and Thr-86-Ile plus Asp-90-Asn, have also beenassociated with FQ resistance in Campylobacter (54). The mutation of Asn-203-Ser is alsoknown to confer FQ resistance along with the Thr-86-Ile mutation (55). Ser-22-Gly hasnot been linked to FQ resistance in Campylobacter, and there was no evidence thatAsn-203-Ser and Arg-285-Lys alone were associated with the FQ resistance phenotype(15). In this study, all examined FQr Campylobacter isolates harbored the Thr-86-Ilemutation in GyrA, while one isolate also carried the Ser-22-Gly, Asn-203-Ser, andArg-285-Lys mutations, consistent with the known role of the Thr-86-Ile change (aloneor along with other mutations) in CIP resistance.

In summary, we observed high prevalences of tetracycline-resistant Campylobacterand FQr C. coli in commercial sheep operations, regardless of in-feed medication withtetracycline. Although the medication increased the total Campylobacter CFU counts inthe sheep gallbladder, it did not affect overall prevalence, genotypes, or antimicrobialsusceptibility profiles of the Campylobacter isolates under the conditions employed inthis study. The sheep harbored predominant genotypes of C. jejuni (ST2862) and C. coli(ST902). Of particular note, the FQr C. coli clones in the sheep (both ST902 and ST1068)were genetically linked to the isolates previously identified in cattle, suggesting thepossibility of dynamic transmission of FQr C. coli between sheep and cattle. However,there are several limitations in this study. First, the work was a controlled laboratorytreatment study, and the preexisting high prevalence of tetracycline-resistant Campy-lobacter strains limited our ability to analyze the effect of the in-feed medication ondevelopment of tetracycline resistance. Second, the sheep were derived only from twocommercial farms, and the sample sizes were not particularly large. Thus, the findingsmay not represent the overall situation with sheep Campylobacter in the United States.Despite these limitations, the findings provide new and useful information on sheepCampylobacter. Since ruminants are important reservoirs for Campylobacter and are a

Xia et al. Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 10

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

significant source of foodborne campylobacteriosis in humans, enhanced efforts shouldbe directed toward the development of interventions to reduce prevalence and trans-mission of FQr Campylobacter in sheep and cattle.

MATERIALS AND METHODSSample collection and Campylobacter isolation. Two separate trials were conducted, and each

involved 80 feeder lambs, with 40 each in the feed-medicated group and feed-nonmedicated group.These sheep were purchased from two commercial farms and then housed in the laboratory animalfacility at Iowa State University during the treatment study. Trial 1 was conducted using animals fromfarm 1, while trial 2 was performed using animals from farm 2. The two farms are in the Midwest regionand are geographically separated by approximately 86 miles. The ages of the sheep at enrollment varied,but all were spring-born feeder lambs and were approximately 2 months old. Both source farms raisesheep in a semi-intensive management program with outdoor access. Treatment of sheep with FQantibiotics is expressly prohibited in the United States, and there was no history of use of theseantibiotics on these farms.

In each trial, the animals were randomly assigned into the feed-medicated and nonmedicatedgroups. Chlortetracycline (Aureomycin; Zoetis) was used for the feed medication, and the medication wasconducted to determine the effect of the antibiotic on prevention and control of bacterial pneumoniain feedlot lambs. The antibiotic was fed extralabel (350 mg/head/day; 160 mg/kg body weight or160 ppm in feed) in accordance with FDA/Center for Veterinary Medicine (CVM) Guidance 615.115.Regardless of the feed medication, clinically sick animals (19 in each group for trial 1; 17 and 18 in eachgroup, respectively, for trial 2) received a chlortetracycline injection (9 mg/lb), and the injection wasrepeated once in 72 h if needed. The treated sheep remained in their respective groups.

Trial 1 was performed from 21 June 2017 to 6 July 2017, while trial 2 was carried out from 13 July2017 to 26 July 2017 (Table 1). Fecal samples were collected at the beginning of the study and once aweek thereafter. Bile and gallbladder samples were collected at necropsy. The samples were thencultured for Campylobacter using Mueller-Hinton (MH) agar plates (Difco) with selective antimicrobialsand growth supplements (SR084E and SR117E; Oxoid). The plates were incubated at 42°C for 48 h undermicroaerobic conditions (85% N2, 5% O2, and 10% CO2). Up to three Campylobacter-like colonies fromeach sample were subcultured onto MH agar plates, and the pure cultures were stored in glycerol stocksat �80°C.

Enumeration of Campylobacter in bile and gallbladder mucosa. In addition to isolation ofCampylobacter from individual bile samples as described above, we also determined the total Campy-lobacter CFU in bile and gallbladder mucosa. For this purpose, in the two trials, bile and gallbladdersamples were randomly collected from sheep at necropsy. In total, 37 and 35 bile/gallbladder mucosalsamples were selected in the two trials from the feed-nonmedicated and feed-medicated groups,respectively. For each group, the sheep were housed in 4 separate pens, and the collected bile from eachgallbladder (5 to 15 ml) was pooled based on treatment group and pen number. In total, 4 pools wereobtained per group in each trial. For collection of gallbladder mucosal samples, 5 ml of phosphate-buffered saline (PBS) was used to wash any residual bile off each gallbladder mucosa. Then, a sterile razorblade was used to scrape mucosa from each gallbladder onto a bile-free weigh boat. Next, the blade waswashed with PBS to rinse any mucosa on the blade onto the weigh boat, which then was rinsed withroughly 2 ml of PBS, and the collected gallbladder mucosa was decanted into a 15-ml conical tube. Thefinal volume was adjusted to 3 ml, and the 15-ml conical tube was vortexed vigorously prior to culturefor Campylobacter.

For both the bile and gallbladder mucosal samples, a 10-fold dilution series was performed using1.5-ml snap cap tubes filled with 900 �l of MH broth. Each dilution was spread onto MH agar platescontaining selective supplement (SR084E and SR117E; Oxoid), and standard culture conditions forCampylobacter were utilized as for the fecal samples (described above). After culturing, the Campylo-bacter CFU were counted for each sample and the log10 averages were computed for both bile andgallbladder mucosa. The detection limit of the plating method was 100 CFU/ml of bile or gallbladdermucosal suspension. All of the 16 pooled bile samples demonstrated visually pure growth of Campylo-bacter spp. and were utilized in the final calculations. For the gallbladder mucosal samples, 4 in thenonmedicated group and 9 in the medicated group were not usable because of high backgroundbacterial contamination after culturing. The remaining plates were visually determined to have puregrowth of Campylobacter spp. and thus were used in the final calculations. In total, Campylobacter CFUcounts were obtained from 33 and 26 gallbladder mucosal samples in the nonmedicated and medicatedgroups, respectively. Representative colonies from each group were selected and subjected to speciesconfirmation and identification (see “Bacterial species identification,” below).

Bacterial species identification. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (Bruker) was used for bacterial species identification. All isolates were identifiedto the species level. Sample preparation and analysis were done as described previously (56). Massspectra were acquired and analyzed using a microflex LT mass spectrometer (Bruker Daltonics) incombination with research use only version of the MALDI Biotyper Compass software 4.1 and thereference database MBT 7311 MSP Library (no. 1829023) at Iowa State University. Data were interpretedin accordance with the manufacturer’s (Bruker Daltonics) standard criteria, as follows: (i) high-confidenceidentification when the score was between 2.00 and 3.00, (ii) low-confidence identification when thescore was between 1.70 and 1.99, and (iii) no organism identification possible when the score was 1.69and lower.

Campylobacter in Sheep Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 11

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

C. jejuni clone SA detection. PCR was used for identifying C. jejuni clone SA. DNA template wasextracted from Campylobacter colonies using single-cell lysis buffer (57). Primers specific for C. jejuniclone SA were designed as described previously (58). C. jejuni IA3902, a clinical isolate of clone SA, wasused as the positive control, while a reaction without template DNA served as a negative control.

Antimicrobial susceptibility testing. A total of 236 C. jejuni and 101 C. coli nonduplicate isolatesfrom fecal samples were tested for antimicrobial susceptibility. Those isolates were representatives ofindividual animals at different sampling times. Also, 98 C. jejuni and 2 C. coli nonduplicate isolates (asingle isolate from each animal) from bile samples were included in the susceptibility testing. The MICsof nine antibiotics were determined using a standard broth microdilution method as recommended byCLSI and the National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS) (59–61).The tested ranges and breakpoints of the nine antibiotics are listed in Table S1. Commercially availableSensititre Campylobacter plates (Thermo Fisher Scientific, Waltham, MA) were used for the tests. The nineantibiotics were azithromycin (AZI), ciprofloxacin (CIP), erythromycin (ERY), gentamicin (GEN), tetracycline(TET), florfenicol (FFN), nalidixic acid (NAL), telithromycin (TEL), and clindamycin (CLI). After incubation ina microaerobic environment for 24 h at 42°C, the results were interpreted. For each isolate, the MIC valuewas set as the lowest antimicrobial concentration at which no bacterial growth was observed. Theantimicrobial resistance breakpoints were chosen according to the standards established by CLSI forbacteria isolated from animals (59–61). C. jejuni ATCC 33560 and C. coli ATCC 33559 were used as qualitycontrol strains.

PFGE and MLST typing of Campylobacter isolates. Representative Campylobacter isolates wereanalyzed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PFGEanalysis of the macrorestriction fragment patterns of genomic DNA using KpnI enzyme was performedfollowing the CDC’s standardized PulseNet protocol for Campylobacter, with minor modifications (23).Briefly, fresh cultures of Campylobacter were embedded in 1% Seakem Gold agarose (Fisher Scientific,Fair Lawn, NJ) and lysed with proteinase K for 30 min at 50°C in a water bath shaker. The gel plugs weredigested with KpnI for 4 h at 37°C. Digested plugs were embedded into 1% agarose and separated byelectrophoresis in 0.5 � Tris-borate-EDTA (TBE) buffer (Promega) at 14°C for 18 h using a Chef Mapperelectrophoresis system (Bio-Rad, Hercules, CA). The gels were stained with ethidium bromide for 30 minand then photographed using a digital imager (Alpha Innotech, Santa Clara, CA). The PFGE patterns wereanalyzed by GelCompare II v.6.5 software (Applied Maths, Kortrijk, Belgium) using the Dice similaritycoefficient and unweighted-pair group method with arithmetic averages (UPGMA) with 0.5% optimiza-tion and 1.5% position tolerance. A Lambda DNA ladder (Bio-Rad) was used as the molecular size marker.PFGE patterns with �90% similarity to each other were defined as a cluster.

To further confirm the genetic diversity of the isolates, MLST was performed for selected represen-tative isolates of different PFGE patterns, as described previously (62). The seven housekeeping geneswere amplified and sequenced using the primers recommended by the Campylobacter MLST website(https://pubmlst.org/campylobacter/), which was developed by Keith Jolley and Man-Suen Chan at theUniversity of Oxford (63). All PCR products were purified using the QIAquick PCR purification kit (Qiagen,Hilden, Germany) and then sequenced at the DNA Core Facility of Iowa State University using an AppliedBiosystems 3730xl DNA analyzer. The sequences were submitted to the MLST database to determineallele numbers and STs. The new alleles and STs were submitted to the database and were assigned newnumbers.

gyrA mutation determination. In Campylobacter, FQ resistance is conferred by point mutations inthe gyrA gene in conjunction with the CmeABC efflux pump (64). To confirm the mechanism of FQresistance in this study, four FQr C. jejuni isolates and eight FQr C. coli isolates of different PFGE and MLSTtypes were selected for determination of the point mutations in the quinolone resistance-determiningregion (QRDR) of gyrA. The primers GyrAF1 (5=-CAACTGGTTCTAGCCTTTTG-3=) and GyrAR1 (5=-AATTTCACTCATAGCCTCACG-3=) were used for C. jejuni isolates, while GyrAF2 (5=-TTATTTAGATTATTCTATGAGCGT-3=) and GyrAR2 (5=-CTTGAGTTCGATTACAACAC-3=) were used for C. coli isolates, as described previously(15). All PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany)and then sequenced at the DNA Core Facility of Iowa State University using an Applied Biosystems 3730xlDNA analyzer.

Statistical analysis. Chi-square and Fisher’s exact tests were used to compare the prevalences andthe antimicrobial resistance rates of the Campylobacter isolates from different samples (fecal and bilesamples) and different groups (feed-nonmedicated and feed-medicated groups). An unpaired t test wasused to compare the average Campylobacter CFU/ml results from the bile and gallbladder mucosalsamples between the nonmedicated and medicated groups in two trials. The data were analyzed usingGraphPad (Prism). P values of �0.05 were deemed to be statistically significant.

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/AEM

.00008-19.SUPPLEMENTAL FILE 1, PDF file, 0.4 MB.

ACKNOWLEDGMENTSThis work was supported by the Frank Ramsey Endowed Chair Fund to Iowa State

University and the USDA Minor Use Animal Drug Program. Jing Xia was supported bya fellowship from the Graduate Student Overseas Study Program of South China

Xia et al. Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 12

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

Agricultural University (grant 2017LHPY030). Jinji Pang was supported by the OneHealth Program for Veterinary Students funded by Zoetis. Lining Xia and Xiaoping Mawere supported by scholarship funds from China Scholarship Council (CSC no.201408655097 and 201606915021, respectively). Melda M. Ocal was supported by theInternational Postdoctoral Research Scholarship Program (no. 2219) of the Scientificand Technological Research Council of Turkey.

REFERENCES1. Sahin O, Yaeger M, Wu Z, Zhang Q. 2017. Campylobacter-associated

diseases in animals. Annu Rev Anim Biosci 5:21– 42. https://doi.org/10.1146/annurev-animal-022516-022826.

2. Kirk MD, Pires SM, Black RE, Caipo M, Crump JA, Devleesschauwer B,Dopfer D, Fazil A, Fischer-Walker CL, Hald T, Hall AJ, Keddy KH, Lake RJ,Lanata CF, Torgerson PR, Havelaar AH, Angulo FJ. 2015. World HealthOrganization estimates of the global and regional disease burden of 22foodborne bacterial, protozoal, and viral diseases, 2010: a data synthesis.PLoS Med 12:e1001921. https://doi.org/10.1371/journal.pmed.1001921.

3. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL,Jones JL, Griffin PM. 2011. Foodborne illness acquired in the UnitedStates—major pathogens. Emerg Infect Dis 17:7–15. https://doi.org/10.3201/eid1701.P11101.

4. Marder EP, Griffin PM, Cieslak PR, Dunn J, Hurd S, Jervis R, Lathrop S,Muse A, Ryan P, Smith K, Tobin-D’Angelo M, Vugia DJ, Holt KG, WolpertBJ, Tauxe R, Geissler AL. 2018. Preliminary incidence and trends ofinfections with pathogens transmitted commonly through food – Food-borne Disease Active Surveillance Network, 10 U. S. sites, 2006 –2017.MMWR Morb Mortal Wkly Rep 67:324 –328. https://doi.org/10.15585/mmwr.mm6711a3.

5. Kaakoush NO, Castano-Rodriguez N, Mitchell HM, Man SM. 2015. Globalepidemiology of Campylobacter infection. Clin Microbiol Rev 28:687–720. https://doi.org/10.1128/CMR.00006-15.

6. Pedersen SK, Wagenaar JA, Vigre H, Roer L, Mikoleit M, Cawthorne AL,Aarestrup FM, Hendriksen RS. 2018. Proficiency of WHO Global Food-borne Infections Network External Quality Assurance System partici-pants in the identification and susceptibility testing of thermotolerantCampylobacter spp. from 2003 to 2012. J Clin Microbiol 56:e01066-18.https://doi.org/10.1128/JCM.01066-18.

7. Sproston EL, Wimalarathna HML, Sheppard SK. 2018. Trends in fluoro-quinolone resistance in Campylobacter. Microb Genom 4:e000198.https://doi.org/10.1099/mgen.0.000198.

8. Tang Y, Fang L, Xu C, Zhang Q. 2017. Antibiotic resistance trends andmechanisms in the foodborne pathogen, Campylobacter. Anim HealthRes Rev 18:87–98. https://doi.org/10.1017/S1466252317000135.

9. Shen Z, Wang Y, Zhang Q, Shen J. 2018. Antimicrobial resistance inCampylobacter spp. Microbiol Spectr 6:ARBA-0013-2017. https://doi.org/10.1128/microbiolspec.ARBA-0013-2017.

10. Oporto B, Esteban JI, Aduriz G, Juste RA, Hurtado A. 2007. Prevalenceand strain diversity of thermophilic campylobacters in cattle, sheep andswine farms. J Appl Microbiol 103:977–984. https://doi.org/10.1111/j.1365-2672.2007.03328.x.

11. Mullner P, Shadbolt T, Collins-Emerson JM, Midwinter AC, Spencer SE,Marshall J, Carter PE, Campbell DM, Wilson DJ, Hathaway S, Pirie R,French NP. 2010. Molecular and spatial epidemiology of humancampylobacteriosis: source association and genotype-related riskfactors. Epidemiol Infect 138:1372–1383. https://doi.org/10.1017/S0950268809991579.

12. Sahin O, Kassem II, Shen Z, Lin J, Rajashekara G, Zhang Q. 2015. Cam-pylobacter in poultry: ecology and potential interventions. Avian Dis59:185–200. https://doi.org/10.1637/11072-032315-Review.

13. Anderson RC, Harvey RB, Wickersham TA, MacDonald JC, Ponce CH,Brown M, Pinchak WE, Osterstock JB, Krueger N, Nisbet DJ. 2014. Effectof distillers feedstuffs and lasalocid on Campylobacter carriage in feedlotcattle. J Food Prot 77:1968 –1975. https://doi.org/10.4315/0362-028X.JFP-14-169.

14. Bianchini V, Luini M, Borella L, Parisi A, Jonas R, Kittl S, Kuhnert P. 2014.Genotypes and antibiotic resistances of Campylobacter jejuni isolatesfrom cattle and pigeons in dairy farms. Int J Environ Res Public Health11:7154 –7162. https://doi.org/10.3390/ijerph110707154.

15. Tang Y, Sahin O, Pavlovic N, LeJeune J, Carlson J, Wu Z, Dai L, Zhang Q.2017. Rising fluoroquinolone resistance in Campylobacter isolated from

feedlot cattle in the United States. Sci Rep 7:494. https://doi.org/10.1038/s41598-017-00584-z.

16. Scott L, Menzies P, Reid-Smith RJ, Avery BP, McEwen SA, Moon CS, BerkeO. 2012. Antimicrobial resistance in Campylobacter spp. isolated fromOntario sheep flocks and associations between antimicrobial use andantimicrobial resistance. Zoonoses Public Health 59:294 –301. https://doi.org/10.1111/j.1863-2378.2011.01450.x.

17. Wu Z, Sippy R, Sahin O, Plummer P, Vidal A, Newell D, Zhang Q. 2014.Genetic diversity and antimicrobial susceptibility of Campylobacterjejuni isolates associated with sheep abortion in the United Statesand Great Britain. J Clin Microbiol 52:1853–1861. https://doi.org/10.1128/JCM.00355-14.

18. Stanley K, Jones K. 2003. Cattle and sheep farms as reservoirs of Cam-pylobacter. J Appl Microbiol 94:104S–113S. https://doi.org/10.1046/j.1365-2672.94.s1.12.x.

19. Thepault A, Rose V, Quesne S, Poezevara T, Beven V, Hirchaud E, TouzainF, Lucas P, Meric G, Mageiros L, Sheppard SK, Chemaly M, Rivoal K. 2018.Ruminant and chicken: important sources of campylobacteriosis inFrance despite a variation of source attribution in 2009 and 2015. SciRep 8:9305. https://doi.org/10.1038/s41598-018-27558-z.

20. Tyson GH, Tate HP, Abbott J, Tran TT, Kabera C, Crarey E, Young S,McDermott PF, Sprague G, Campbell M, Adeyemo O, Browne-Silva J,Myers M, Thitaram S, Zhao S. 2016. Molecular subtyping and sourceattribution of Campylobacter isolated from food animals. J Food Prot79:1891–1897. https://doi.org/10.4315/0362-028X.JFP-16-195.

21. de Haan CPA, Kivistö RI, Hakkinen M, Corander J, Hänninen M-L. 2010.Multilocus sequence types of Finnish bovine Campylobacter jejuni iso-lates and their attribution to human infections. BMC Microbiol 10:200.https://doi.org/10.1186/1471-2180-10-200.

22. Smith JL. 2002. Campylobacter jejuni infection during pregnancy:longterm consequences of associated bacteremia, Guillain-Barre syn-drome, and reactive arthritis. J Food Prot 65:696 –708. https://doi.org/10.4315/0362-028X-65.4.696.

23. Sahin O, Plummer PJ, Jordan DM, Sulaj K, Pereira S, Robbe-Austerman S,Wang L, Yaeger MJ, Hoffman LJ, Zhang Q. 2008. Emergence of atetracycline-resistant Campylobacter jejuni clone associated with out-breaks of ovine abortion in the United States. J Clin Microbiol 46:1663–1671. https://doi.org/10.1128/JCM.00031-08.

24. Wu Z, Periaswamy B, Sahin O, Yaeger M, Plummer P, Zhai W, Shen Z, DaiL, Chen SW, Zhang Q. 2016. Point mutations in the major outer mem-brane protein drive hypervirulence of a rapidly expanding clone ofCampylobacter jejuni. Proc Natl Acad Sci U S A 113:10690 –10695. https://doi.org/10.1073/pnas.1605869113.

25. Sahin O, Fitzgerald C, Stroika S, Zhao S, Sippy RJ, Kwan P, Plummer PJ,Han J, Yaeger MJ, Zhang Q. 2012. Molecular evidence for zoonotictransmission of an emergent, highly pathogenic Campylobacter jejuniclone in the United States. J Clin Microbiol 50:680 – 687. https://doi.org/10.1128/JCM.06167-11.

26. Sanad YM, Closs GJ, Kumar A, LeJeune JT, Rajashekara G. 2013. Molecularepidemiology and public health relevance of Campylobacter isolatedfrom dairy cattle and European starlings in Ohio, USA. Foodborne Pat-hog Dis 10:229 –236. https://doi.org/10.1089/fpd.2012.1293.

27. Smith AB, Renter DG, Cernicchiaro N, Shi X, Nickell JS, Keil DJ, NagarajaTG. 2017. A randomized trial to assess the effect of fluoroquinolonemetaphylaxis on the fecal prevalence and quinolone susceptibilities ofSalmonella and Campylobacter in feedlot cattle. Foodborne Pathog Dis14:600 – 607. https://doi.org/10.1089/fpd.2017.2282.

28. Noormohamed A, Fakhr MK. 2013. A higher prevalence rate of Campy-lobacter in retail beef livers compared to other beef and pork meat cuts.Int J Environ Res Public Health 10:2058 –2068. https://doi.org/10.3390/ijerph10052058.

29. Acik MN, Cetinkaya B. 2006. Heterogeneity of Campylobacter jejuni and

Campylobacter in Sheep Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 13

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from

Campylobacter coli strains from healthy sheep. Vet Microbiol 115:370 –375. https://doi.org/10.1016/j.vetmic.2006.02.014.

30. Ertas HB, Ozbey G, Kiliç A, Muz A. 2003. Isolation of Campylobacter jejuni andCampylobacter coli from the gall bladder samples of sheep and identifica-tion by polymerase chain reaction. J Vet Med B Infect Dis Vet Public Health50:294–297. https://doi.org/10.1046/j.1439-0450.2003.00678.x.

31. Lin J, Michel LO, Zhang Q. 2002. CmeABC functions as a multidrug effluxsystem in Campylobacter jejuni. Antimicrob Agents Chemother 46:2124 –2131. https://doi.org/10.1128/AAC.46.7.2124-2131.2002.

32. Lin J, Sahin O, Michel LO, Zhang Q. 2003. Critical role of multidrug effluxpump CmeABC in bile resistance and in vivo colonization of Campylo-bacter jejuni. Infect Immun 71:4250 – 4259. https://doi.org/10.1128/IAI.71.8.4250-4259.2003.

33. Lin J, Cagliero C, Guo B, Barton YW, Maurel MC, Payot S, Zhang Q. 2005.Bile salts modulate expression of the CmeABC multidrug efflux pump inCampylobacter jejuni. J Bacteriol 187:7417–7424. https://doi.org/10.1128/JB.187.21.7417-7424.2005.

34. Lei HT, Shen Z, Surana P, Routh MD, Su CC, Zhang Q, Yu EW. 2011.Crystal structures of CmeR-bile acid complexes from Campylobacterjejuni. Protein Sci 20:712–723. https://doi.org/10.1002/pro.602.

35. Tang Y, Meinersmann RJ, Sahin O, Wu Z, Dai L, Carlson J, Lawrence JP,Genzlinger L, LeJeune JT, Zhang Q. 2017. Wide but variable distributionof a hypervirulent Campylobacter jejuni clone in beef and dairy cattle inthe United States. Appl Environ Microbiol 83:e01425-17. https://doi.org/10.1128/AEM.01425-17.

36. Stone DM, Chander Y, Bekele AZ, Goyal SM, Hariharan H, Tiwari K,Chikweto A, Sharma R. 2014. Genotypes, antibiotic resistance, and ST-8genetic clone in Campylobacter isolates from sheep and goats in Gre-nada. Vet Med Int 2014:212864. https://doi.org/10.1155/2014/212864.

37. Colles FM, Jones K, Harding RM, Maiden M. 2003. Genetic diversity ofCampylobacter jejuni isolates from farm animals and the farm environ-ment. Appl Environ Microbiol 69:7409 –7413. https://doi.org/10.1128/AEM.69.12.7409-7413.2003.

38. Grove-White DH, Leatherbarrow AJ, Cripps PJ, Diggle PJ, French NP.2011. Molecular epidemiology and genetic diversity of Campylobacterjejuni in ruminants. Epidemiol Infect 139:1661–1671. https://doi.org/10.1017/S0950268810002736.

39. Zhang M, Gu Y, He L, Ran L, Xia S, Han X, Li H, Zhou H, Cui Z, Zhang J.2010. Molecular typing and antimicrobial susceptibility profiles of Cam-pylobacter jejuni isolates from north China. J Med Microbiol 59:1171–1177. https://doi.org/10.1099/jmm.0.022418-0.

40. Rotariu O, Dallas JF, Ogden ID, MacRae M, Sheppard SK, Maiden MC,Gormley FJ, Forbes KJ, Strachan NJ. 2009. Spatiotemporal homogeneityof Campylobacter subtypes from cattle and sheep across northeasternand southwestern Scotland. Appl Environ Microbiol 75:6275– 6281.https://doi.org/10.1128/AEM.00499-09.

41. Sheppard SK, Dallas JF, MacRae M, McCarthy ND, Sproston EL, GormleyFJ, Strachan NJ, Ogden ID, Maiden MC, Forbes KJ. 2009. Campylobactergenotypes from food animals, environmental sources and clinical dis-ease in Scotland 2005/6. Int J Food Microbiol 134:96 –103. https://doi.org/10.1016/j.ijfoodmicro.2009.02.010.

42. Miller WG, Englen MD, Kathariou S, Wesley IV, Wang G, Pittenger-Alley L,Siletz RM, Muraoka W, Fedorka-Cray PJ, Mandrell RE. 2006. Identificationof host-associated alleles by multilocus sequence typing of Campylobac-ter coli strains from food animals. Microbiology 152:245–255. https://doi.org/10.1099/mic.0.28348-0.

43. Abley MJ, Wittum TE, Funk JA, Gebreyes WA. 2012. Antimicrobial sus-ceptibility, pulsed-field gel electrophoresis, and multi-locus sequencetyping of Campylobacter coli in swine before, during, and after theslaughter process. Foodborne Pathog Dis 9:506 –512. https://doi.org/10.1089/fpd.2011.1053.

44. Quintana-Hayashi MP, Thakur S. 2012. Phylogenetic analysis revealscommon antimicrobial resistant Campylobacter coli population inantimicrobial-free (ABF) and commercial swine systems. PLoS One7:e44662. https://doi.org/10.1371/journal.pone.0044662.

45. Litrup E, Torpdahl M, Nielsen EM. 2007. Multilocus sequence typingperformed on Campylobacter coli isolates from humans, broilers, pigsand cattle originating in Denmark. J Appl Microbiol 103:210 –218.https://doi.org/10.1111/j.1365-2672.2006.03214.x.

46. Vinueza-Burgos C, Wautier M, Martiny D, Cisneros M, Van Damme I,De Zutter L. 2017. Prevalence, antimicrobial resistance and geneticdiversity of Campylobacter coli and Campylobacter jejuni in Ecuador-ian broilers at slaughter age. Poult Sci 96:2366 –2374. https://doi.org/10.3382/ps/pew487.

47. Kittl S, Heckel G, Korczak BM, Kuhnert P. 2013. Source attribution ofhuman Campylobacter isolates by MLST and fla-typing and associationof genotypes with quinolone resistance. PLoS One 8:e81796. https://doi.org/10.1371/journal.pone.0081796.

48. Sproston EL, Ogden ID, MacRae M, Dallas JF, Sheppard SK, Cody AJ,Colles FM, Wilson MJ, Forbes KJ, Strachan NJ. 2011. Temporal variationand host association in the Campylobacter population in a longitudinalruminant farm study. Appl Environ Microbiol 77:6579 – 6586. https://doi.org/10.1128/AEM.00428-11.

49. Cha W, Mosci RE, Wengert SL, Venegas Vargas C, Rust SR, Bartlett PC,Grooms DL, Manning SD. 2017. Comparing the genetic diversity andantimicrobial resistance profiles of Campylobacter jejuni recovered fromcattle and humans. Front Microbiol 8:818. https://doi.org/10.3389/fmicb.2017.00818.

50. Ragimbeau C, Schneider F, Losch S, Even J, Mossong J. 2008. Multilocussequence typing, pulsed-field gel electrophoresis, and fla short variableregion typing of clonal complexes of Campylobacter jejuni strains ofhuman, bovine, and poultry origins in Luxembourg. Appl Environ Mi-crobiol 74:7715–7722. https://doi.org/10.1128/AEM.00865-08.

51. Noormohamed A, Fakhr MK. 2014. Molecular typing of Campylobacterjejuni and Campylobacter coli isolated from various retail meats by MLSTand PFGE. Foods 3:82–93. https://doi.org/10.3390/foods3010082.

52. Ge B, McDermott PF, White DG, Meng J. 2005. Role of efflux pumps andtopoisomerase mutations in fluoroquinolone resistance in Campylobac-ter jejuni and Campylobacter coli. Antimicrob Agents Chemother 49:3347–3354. https://doi.org/10.1128/AAC.49.8.3347-3354.2005.

53. Wang Y, Huang WM, Taylor DE. 1993. Cloning and nucleotide se-quence of the Campylobacter jejuni gyrA gene and characterization ofquinolone resistance mutations. Antimicrob Agents Chemother 37:457– 463. https://doi.org/10.1128/AAC.37.3.457.

54. Payot S, Bolla JM, Corcoran D, Fanning S, Megraud F, Zhang Q. 2006.Mechanisms of fluoroquinolone and macrolide resistance in Campylo-bacter spp. Microbes Infect 8:1967–1971. https://doi.org/10.1016/j.micinf.2005.12.032.

55. Corcoran D, Quinn T, Cotter L, Fanning S. 2005. Relative contribution oftarget gene mutation and efflux to varying quinolone resistance in IrishCampylobacter isolates. FEMS Microbiol Lett 253:39 – 46. https://doi.org/10.1016/j.femsle.2005.09.019.

56. Schulthess B, Bloemberg GV, Zbinden A, Mouttet F, Zbinden R, BottgerEC, Hombach M. 2016. Evaluation of the Bruker MALDI Biotyper foridentification of fastidious Gram-negative rods. J Clin Microbiol 54:543–548. https://doi.org/10.1128/JCM.03107-15.

57. Olah PA, Doetkott C, Fakhr MK, Logue CM. 2006. Prevalence of theCampylobacter multi-drug efflux pump (CmeABC) in Campylobacter spp.isolated from freshly processed turkeys. Food Microbiol 23:453– 460.https://doi.org/10.1016/j.fm.2005.06.004.

58. Luo Y, Sahin O, Dai L, Sippy R, Wu Z, Zhang Q. 2012. Development of aloop-mediated isothermal amplification assay for rapid, sensitive andspecific detection of a Campylobacter jejuni clone. J Vet Med Sci 74:591–596. https://doi.org/10.1292/jvms.11-0462.

59. Clinical and Laboratory Standards Institute. 2013. Performance standardsfor antimicrobial disk and dilution susceptibility tests for bacteria iso-lated from animals, approved standard— 4th ed. CLSI document VET01-A4. Clinical and Laboratory Standards Institute, Wayne, PA.

60. Ge B, Wang F, Sjolund-Karlsson M, McDermott PF. 2013. Antimicrobialresistance in Campylobacter: susceptibility testing methods and resis-tance trends. J Microbiol Methods 95:57– 67. https://doi.org/10.1016/j.mimet.2013.06.021.

61. Clinical and Laboratory Standards Institute. 2016. Methods for antimi-crobial dilution and disk susceptibility testing of infrequently isolated orfastidious bacteria, 3rd ed. CLSI guideline M45. Clinical and LaboratoryStandards Institute, Wayne, PA.

62. Dingle KE, Colles FM, Wareing DR, Ure R, Fox AJ, Bolton FE, Bootsma HJ,Willems RJ, Urwin R, Maiden MC. 2001. Multilocus sequence typingsystem for Campylobacter jejuni. J Clin Microbiol 39:14 –23. https://doi.org/10.1128/JCM.39.1.14-23.2001.

63. Jolley KA, Chan MS, Maiden MC. 2004. mlstdbNet – distributed multi-locussequence typing (MLST) databases. BMC Bioinformatics 5:86. https://doi.org/10.1186/1471-2105-5-86.

64. Zhang Q, Zhang Q, Lin J, Pereira S. 2003. Fluoroquinolone-resistantCampylobacter in animal reservoirs: dynamics of development, resis-tance mechanisms and ecological fitnes. Anim Health Res Rev 4:63–71.https://doi.org/10.1079/AHR200356.

Xia et al. Applied and Environmental Microbiology

June 2019 Volume 85 Issue 11 e00008-19 aem.asm.org 14

on February 25, 2021 by guest

http://aem.asm

.org/D

ownloaded from