In vitro antimicrobial susceptibility, genetic diversity and prevalence of UDP-glucose 4-epimerase...

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FOODMICROBIOLOGY

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doi:10.1016/j.fm

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Food Microbiology 23 (2006) 379–392

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In vitro antimicrobial susceptibility, genetic diversity and prevalenceof UDP-glucose 4-epimerase (galE) gene in Campylobacter coli and

Campylobacter jejuni from Turkey production facilities

Rajesh Nayak�, Tabitha Stewart1, Mohamed Nawaz, Carl Cerniglia

US Food and Drug Administration, National Center for Toxicological Research, Division of Microbiology, 3900 NCTR Road, Jefferson, AR 72079,

USA

Received 16 February 2005; received in revised form 26 April 2005; accepted 26 April 2005

Available online 5 July 2005

Abstract

This study evaluated the genetic diversity of multi-drug resistant Campylobacter jejuni (n ¼ 44) and C. coli (n ¼ 30) isolated from

18 turkey houses. Antimicrobial resistances to ampicillin, ciprofloxacin and nalidixic acid were higher (Po0:05) in C. coli than in C.

jejuni strains. PCR analysis indicated that 82% of total isolates tested, including 91% of C. jejuni and 70% of C. coli tested positive

for a 496-bp UDP-glucose 4-epimerase (galE) gene. The diversity of isolates was mapped by antibiogram, SmaI-PFGE and flaA-

RFLP typing methods using the discriminatory index (DI). RFLP was more suitable in discriminating C. coli (DI ¼ 0.895) than

PFGE (DI ¼ 0.816) or antibiogram profile (DI ¼ 0.552), while either PFGE (DI ¼ 0.941) or RFLP (DI ¼ 0.942) could be used in

discriminating C. jejuni strains. The combined PFGE and antibiogram dendrogram had the highest DI for both C. coli (0.910) and

C. jejuni (0.968), suggesting that a combination of typing methods is more useful in examining the diverse Campylobacter population

on turkey farms.

Published by Elsevier Ltd.

Keywords: Campylobacter; Genotyping; Antimicrobial resistance; Turkey; PCR

1. Introduction

Campylobacteriosis is the most commonly reportedhuman bacterial gastroenteritis in the US (Centers forDisease Control and Prevention, 2001). Campylobacter

coli and C. jejuni are responsible for the majority offoodborne illnesses (Nayak et al., 2003). In addition togastroenteritis, Campylobacter spp. have been recog-nized as the most common infectious agents associatedwith the development of Guillain–Barre syndrome(GBS), an autoimmune disorder of the peripheralnervous system (Nachamkin et al., 1998; Hadden and

front matter Published by Elsevier Ltd.

.2005.04.007

ng author. Tel.: +1870 543 7482;

7307.

ss: [email protected] (R. Nayak).

ess: University of Tennessee Health Science Center,

163, USA.

Gregson, 2001). Nearly 13–49% of the GBS casesmaybe preceded by C. jejuni infections and C. jejuni-enteritis induced an antecedent infection in �30% ofGBS cases (Dingle et al., 2001). Although studies havelinked Campylobacter spp. with the onset of GBS, todate there is no conclusive evidence to associate specificgenotypes with the onset of GBS (Endtz et al., 2000). Inmost cases, C. jejuni, representing the Penner serotypeO:19 and O:41 lineages, has been identified as theetiological agent in Campylobacter infections in patientswith subsequent GBS complications (Kuroki et al.,1993). Approximately one in every 1000 C. jejuni

infections is followed by GBS (Allos, 1997). The galE

gene encoding UDP-glucose 4-epimerase, which cata-lyses the interconversion of UDP-galactose and UDP-glucose, is involved in the synthesis of lipopolysacchar-ide (LPS) in C. jejuni (Fry et al., 2000). N-acetylneur-aminic acid (sialic acid), a core oligosaccharide molecule

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ARTICLE IN PRESSR. Nayak et al. / Food Microbiology 23 (2006) 379–392380

not frequently found in prokaryotes, is part of theCampylobacter LPS outer and inner core regions (Fry etal., 2000). Sialic acid residues, when attached by 2–3linkages to b-D-galactoside resemble gangliosides instructure. This molecular mimicry has been thought toplay a role in GBS (Fry et al., 2000). As galactose isneeded to form the ganglioside-like LPS structures, thegalE gene may be essential for Campylobacter strains toinduce GBS (B. Fry, pers. comm.).

Campylobacter spp. colonize the intestinal tract ofpoultry, and through horizontal transmission, contam-inates other birds and eventually carcasses. IdenticalCampylobacter genotypes are commonly found in flocksraised simultaneously on broiler farms, and severalclones have been shown to persist during successivebroiler-flock rotations (Nadeau et al., 2002). Thesepersistent Campylobacter clones may be transferred toprocessing facilities, and as a result of cross-contamina-tion at the slaughter houses, fresh broiler meat may becontaminated with genotypes that were not originallypresent in the flock (Newell et al., 2001). Poultry meathas been implicated in a large number of campylobac-teriosis cases in humans (Kassenborg et al., 2004). Amajor human risk factor for acquiring Campylobacter

infections could be through handling and/or consump-tion of contaminated turkey meat (Nayak et al., 2003).Studies have shown widespread genetic diversity amongCampylobacter isolates from human and poultry origin(Koenraad et al., 1995; Manning et al., 2003). However,given the opportunity, Campylobacter isolates frompoultry and its environment may have the potential tocause disease in humans, and these bacteria should beconsidered as potential sources of human infection.

Most Campylobacter infections are self-limiting anddo not require antibiotic therapy. However, patientssuffering from severe cases of invasive Campylobacter

enteritis are treated with erythromycin and ciprofloxacin(Nayak et al., 2003). Fluoroquinolones have also beensparingly used by the poultry industry to treat poultryinfections (Federal Register, 2000). However, overuse ofantibiotics by both the veterinary and medical fields hasresulted in the emergence of Campylobacter strains thatare resistant to a wide range of antibiotics (Endtz et al.,1991; Iovine and Blaser, 2004). Reduction of antibiotic-resistant Campylobacter spp. in raw and processedpoultry necessitates comprehensive control at thebreeder farms, hatcheries, and production facilities.Molecular typing methods can be useful in delineatingpossible Campylobacter transmission pathways fromhatcheries to processing plants, and to determinepossible links with clinical outbreak strains. Severalgenetic fingerprinting techniques, such as pulsed-field gelelectrophoresis (PFGE), restriction fragment lengthpolymorphism (RFLP), amplified fragment lengthpolymorphism (AFLP), rapid amplification of poly-morphic DNA (RAPD), flagellin-gene sequencing,

multi-locus sequence typing (MLST) and multi-locusenzyme electrophoresis (MLEE) have been used todiscriminate Campylobacter genotypes (Wassennar andNewell, 2000; Manning et al., 2003; Matsuda et al.,2003).

Poultry litter serves as a major reservoir of bacteria,which may be pathogenic to poultry and humans(Corpet, 1996). The use of litter as a fertilizer, feed orbedding supplement may result in the transfer ofpathogenic bacteria in the environment, detrimentallyaffecting poultry and human health (Kelley et al., 1998).Campylobacter is not pathogenic to poultry; however,the bacteria can be transmitted to humans via fresh meatand cause foodborne outbreaks. Although litter hasbeen found to transmit C. jejuni in susceptible chicksand broiler flocks (Montrose et al., 1984; Payne et al.,1999), there is limited information on the prevalence andepidemiology of Campylobacter spp. in the turkeyproduction environment (Borck et al., 2001; Nayak etal., 2003a). The role of litter as a medium formaintenance of Campylobacter in turkey flocks andtransmission within flocks and to subsequent flocksneeds to be evaluated. This study investigated thegenetic diversity, antimicrobial resistance and preva-lence of the galE gene determinant in C. coli and C.

jejuni strains isolated from turkey litter.

2. Materials and methods

2.1. Sample collection and isolation of Campylobacter

A total of 74 Campylobacter strains were isolatedfrom litter samples from 18 turkey production facilitieslocated within �10-mile radius in northwestern Arkan-sas. The study was conducted from February 2002 toOctober 2003. About 2–3 g of litter samples (woodshavings), preferably mixed with fecal droppings, werecollected around the drinkers and feeders from 4 to 6pens on each turkey farm. Standard biosecuritymeasures were employed before entering the facility.These included the use of disposable, sterile gowns andboots, mask, sterile gloves during sample collection anddisinfection of boots before entering each facility. Littersamples were collected in sterile 50ml Nalgene tubescontaining 20ml of sterile Preston broth (Remel,Lenexa, Kansas, USA). The mixture was shaken andthe tubes were placed in Campy-gas packs anaerobicchamber (Becton Dickinson Microbiology Systems,Sparks, Maridona, USA) containing Pack-Campylomicroaerophilic gas generating sachets (Mitsubishi GasChemical America, Inc., New York, New York, USA).The samples were stored overnight at 4 1C and processedthe next day. Limited access to privately owned turkeyfarms restricted our ability to collect litter samples atregular intervals. On average, samples were collected

ARTICLE IN PRESSR. Nayak et al. / Food Microbiology 23 (2006) 379–392 381

every 4–6 weeks during the 20–22-week grow-out periodof each flock. The samples were collected through athird party in order to avoid conflict of interest with theUS Food and Drug Administration (FDA). Hence,limited information was available on the number offlocks sampled, sources from which Campylobacter wereisolated, flock number, and history of antibiotics used atthese farms with the exception that sarafloxacin wasused on all these farms.

Overnight mixtures of litter sample and Preston brothwere incubated under microaerophilic conditions (89%nitrogen, 5% carbon dioxide and 6% oxygen) at 42 1Cfor 6–8 h. A loopful of the enriched culture was platedon Campy-agar plates (Remel) and incubated at 42 1Cfor an additional 96 h under microaerophilic conditions.For each presumptive positive agar plate, typicalCampylobacter colonies were subcultured, Gramstained, and tested for cell morphology, hippuratehydrolysis, growth at 42 1C, a-hemolysis on sheep bloodagar, motility and production of oxidase and catalase.

2.2. Antimicrobial susceptibility testing

The antibiotics used were chosen as representatives ofthe various classes of antimicrobial agents in commonuse. The antimicrobial susceptibility was determinedusing a disk-diffusion assay (National Committee forClinical Laboratory Standards (NCCLS), 2000). Over-night cultures, grown on trypticase soy broth (optical

Table 1

Antimicrobial susceptibility testing of Campylobacter isolates from turkey p

Antibiotic Disc concentration (mg) Zone diameter interpretationa

Resistant Intermediate

Ampicillin 10 p13 14–16

Ciprofloxacin 5 p15 16–20

Chloramphenicol 30 p12 13–17

Kanamycin 30 p13 14–17

Bacitracin 10 p8 9–12

Streptomycin 10 p11 12–14

SXTd 23.75/1.25 p10 11–15

Gentamicin 10 p12 13–14

Nalidixic acid 30 p13 14–18

Multiple drug resistance

# of Antibiotics

0 — — —

1 — — —

2 — — —

3 — — —

4 — — —

5 — — —

6 — — —

aBased on BBLs Sensi-Discs antimicrobial zone diameter interpretativebExact two-sided P-value based on the permutation distribution of FishercFive percent significance level.dSulfamethoxazole/trimethoprim.

density adjusted to 0.5 McFarland units), were spreadevenly on Mueller-Hinton agar (Difco Laboratories,Detroit, Michigan, USA) and each Campylobacter

isolate was tested with multiple antibiotics listed inTable 1. The plates were incubated at 42 1C for 24 hunder microaerophilic conditions. The zones of inhibi-tion were measured and interpreted as resistant orsensitive according to the manufacturer’s (BD Bios-ciences, Cockeysville, Maryland, USA) guidelines.

2.3. Multiplex PCR for speciating Campylobacter strains

A multiplex PCR assay was used to distinguish C. coli

and C. jejuni (Nayak et al., 2005). The reaction mixtureconsisted of 2.5 ml of bacterial extract, 2.5 ml of 10�BSA buffer (1ml of 10� contained 500 ml of 1 M

Tris–HCl, pH 8.5, 200 ml of 1M KCl, 30 ml of 1 M

MgCl2, 5mg of BSA and 270 ml of deionized water),2.4 ml of 10� dNTP mixture (2.5mM of each dNTP),0.7 ml each of cadF (Campylobacter spp.), ceuE (C. coli )and a gene encoding for oxidoreductase subunit (C.

jejuni) primer mix (25 mM stock concentration), 0.2 ml ofAmpliTaqs DNA polymerase (5U/ml; Applied Biosys-tems, Foster City, California, USA) and deionized waterto a final volume of 25 ml. The reaction mixture wasamplified in a 9700 GeneAmps PCR system (AppliedBiosystems). The following PCR conditions were used:heat denaturation at 94 1C for 4min, 33 cycles withdenaturation at 94 1C for 1min, annealing at 52 1C for

roduction facilities

(mm) C. coli C. jejuni Total Fisher Exact Testb

Sensitive N ¼ 30 N ¼ 44 N ¼ 74

X17 23 (77%) 21 (48%) 44 (59%) 0.016c

X21 18 (60%) 11 (25%) 29 (39%) 0.004c

X18 1 (3%) 1 (2%) 2 (3%) 1.0

X18 4 (13%) 22 (50%) 26 (35%) 0.001c

X13 28 (93%) 39 (89%) 67 (91%) 0.69

X15 1 (3%) 0 (0%) 1 (1%) 0.41

X16 28 (93%) 42 (95%) 70 (95%) 1.0

X15 1 (3%) 0 (0%) 1 (1%) 0.41

X19 20 (67%) 17 (39%) 37 (50%) 0.032c

— 1 (3%) 0 (0%) 1 (1%) 0.41

— 0 (0%) 2 (5%) 2 (3%) 0.51

— 4 (13%) 12 (27%) 16 (22%) 0.25

— 3 (10%) 10 (23%) 13 (18%) 0.22

— 5 (17%) 9 (20%) 14 (19%) 0.77

— 15 (50%) 5 (11%) 20 (27%) o0.001c

— 2 (7%) 6 (14%) 8 (11%) 0.46

chart.

’s conditional probability statistic.


1min and extension at 72 1C for 1min, and finalextension at 72 1C for 5min. C. coli ATCC 33559 andC. jejuni ATCC 29428 were used as positive controls inall PCR reactions.

2.4. Detection of galE gene

Campylobacter isolates were grown on blood agarplates supplemented with 5% sheep blood at 42 1C for72 h under microaerophilic conditions. Cells weresuspended in 300 ml sterile, deionized, glass-distilledwater and heated in a boiling water bath for 10min.The samples were cooled immediately in an ice bath for5–10min and centrifuged at 13,000g for 5min. Thesupernatant was used as a source of template DNA forPCR. The PCR reaction mixture contained 50mM

Tris–HCl (pH 8.5), 20mM KCl, 3mM MgCl2, 0.05%bovine serum albumin, 0.25mM of each dNTP, 0.25 mMof each primer and 0.9U of Taq polymerase (Invitrogen,Carlsbad, California, USA). Following primers wereused to amplify the galE gene (Accession no: Y11648)(F) 50GGA CCA CAA ACT CCC GTT G30; (R) 50ACACTA GGA TCA CCC GCA C30 (Nawaz et al., 2003).The reaction mixture was amplified in a 9700GeneAmps PCR system (Applied Biosystems), usingthe following reaction conditions: one cycle at 95 1C for4min, then 35 cycles at 95 1C for 10 s, 54 1C for 10 s,72 1C for 40 s, and one final extension cycle at 72 1C for4min. Amplified product was purified by the PCRpurification kit (Qiagen, Inc., Valencia, California,USA) and the purified product was digested withHindIII (Invitrogen) at 37 1C for 2 h to confirm thespecificity of the PCR product. A 100-bp ladder(Invitrogen) was run along with the PCR product on a1% agarose gel to size the DNA fragments.

2.5. Pulsed-field gel electrophoresis (PFGE)

The PFGE procedure was carried out using theprotocol described elsewhere (Nayak et al., 2003a).Briefly, bacterial colonies were suspended in 1–2ml ofcell suspension buffer (100mM Tris–HCl and 100mM

EDTA, pH 8.0). The optical density (610 nm) of the cellsuspension was standardized to 1.3–1.4. A 400 ml-aliquotof each cell suspension was mixed with 20 ml ofproteinase K (20mg/ml) and 400 ml of 1% SeaKemGold agarose (BioWhittaker Molecular Applications,Rockland, Maine, USA) prepared in TE buffer (10mM

Tris–HCl and 1mM EDTA, pH 8.0). The bacterium–agarose mixture was dispensed in plug molds andallowed to solidify at room temperature for 10–15min.Each sample plug was transferred to a 50-ml centrifugetube containing 5ml cell lysis buffer (50mM Tris–HCland 50mM EDTA, pH 8.0+1% sodium lauryl sarco-sine). Proteinase K was added to each tube to give a finalconcentration of 0.1mg/ml. The tubes were incubated in

a water bath maintained at 54 1C for 2–3 h with constantagitation (125–150 rpm). After proteolysis, plugs werewashed twice with 10–15ml of preheated (50 1C)distilled water and 4 times with preheated (50 1C) sterileTE buffer for 10–15min each time. The DNA wasdigested with 50U of SmaI (Invitrogen) at 37 1C for 5 h.The DNA fragments were separated on 1% SeaKemGold agarose using the CHEF-Mapper III PFGEsystem (Bio-Rad Laboratories, Hercules, California,USA). The following electrophoretic conditions wereused: initial switch time, 2.16 s; final switch time, 63.8 s;run time, 18 h; angle, 1201; gradient, 6.0V/cm; tris-borate EDTA (TBE) buffer temperature, 14 1C; andramping factor, linear. Low-range PFGE molecularweight markers (New England BioLabs, Beverly,Massachussets, USA) were used to size the DNAfragments.

2.6. Restriction fragment length polymorphism (RFLP)

The PCR mixture contained 2.0 ml of DNA template,12.5 ml of 2� Qiagen Taq PCR Master mix (Qiagen,Inc.) [Tris–HCl, KCl, and (NH4)2SO4 buffer, pH 8.7;3mM MgCl2, and 400 mM of each dNTP, and 0.05U/mlof Taq polymerase], 0.25 ml of each primer mix (25 mMstock concentration) and deionized water to a finalvolume of 25 ml. The following primers were used toamplify the flaA gene (Accession no.: AB103061) (F)50GGA TTT CGT ATT AAC ACA AAT GGT GC (R)50CTG TAG TAA TCT TAA AAC ATT TTG30

(Nayak et al., 2003a). The reaction mixture wasamplified in a 9700 GeneAmps PCR system. Themixture was incubated at 94 1C for 1min, then cycled 35times at 94 1C for 30 s, at 55 1C for 30 s and 72 1C for1.5min, and finally at 72 1C for 5min. A 16-ml aliquot ofthe flagellin gene PCR product (1.7 kb) was digestedwith 2-ml of the restriction enzyme DdeI (10U/ml;Invitrogen) and 2-ml of React-3 incubation buffer for18–20 h. The DNA fragments were separated on a 3.5%agarose gel at 80V in 1� TBE buffer. A 100-bp DNAladder (Invitrogen) was used as a standard for molecularsize determination.

2.7. Gel visualization

Both PFGE and RFLP gels were stained withethidium bromide and visualized under UV illuminationusing a gel documentation system (UVP BioImagingSystems, Upland, California, USA). Images werecropped and the background was filtered to achievemaximum resolution of the banding pattern, using ScionImaging software (Scion Corp., Frederick, Maryland,USA). Images were saved in the TIFF file format forfurther analysis.


2.8. Antibiogram and restriction profile analyses

The genetic relationship among the Campylobacter

strains was analysed using Bionumerics software (Ap-plied Maths, Sint-Martens-Latem, Belgium). The gelimages were normalized on the basis of molecularweight standards used in identifying the DNA fragmentsize. The antibiogram and PFGE or RFLP fingerprintswere identified as allelic clones when the bandingpatterns shared X90% similarity, with no more thantwo to three band differences (Tenover et al., 1995). Thesimilarity matrix and clustering dendrogram type usedfor cluster analyses were calculated using the ‘Dice’band-matching coefficient and the ‘Unweighted PairGroup Method using Arithmetic Averages (UPGMA)’algorithm, respectively (Nayak et al., 2003a). Accordingto De Boer et al. (2000), ‘the Dice coefficient method isbased on comparison of designated band positions, anddivides the number of matching bands between patternsby the total number of bands, thereby emphasizing thematching bands.’ A positional tolerance shift of 1% ofpattern length was allowed between similar bands andan optimization shift of 1% was allowed between anytwo patterns while generating the dendrograms (Nayaket al., 2003a). The dendrogram based on combined(PFGE+antibiogram) typing methods was generatedby multi-component analysis of the band-matching andUPGMA algorithms, using a binary composite data-set.

The single numerical index of discrimination (DI) wasused to discriminate between the typing methods(Hunter and Gaston, 1988). This value determines theprobability that any two unrelated strains sampled froma test population will be placed into different genotypes.The index was derived using the following equation:

D ¼ 1�1

NðN � 1Þ

Xs

j¼1

njðnj � 1Þ,

where ‘N’ is the total number of strains, ‘s’ is the totalnumber of clusters described, and ‘nj’ is the number ofstrains belonging to the ‘jth’ cluster. The selection ofnodes on the dendrograms was based on clusters whichshared X80% similarity with no more than 2–3 band orantibiotic resistance differences between genotypes with-in each node.

3. Results

3.1. Prevalence and antibiotic susceptibility profile

A total of 74 different Campylobacter strains wereisolated from 16% of 460 litter samples. Of the 74isolates, the PCR assay identified 44 C. jejuni and 30 C.

coli strains. Campylobacter isolates were resistant toampicillin (59%), ciprofloxacin (39%), kanamycin

(35%), bacitracin (91%), sulfamethoxazole:trimetho-prim (SXT; 95%) and nalidixic acid (50%; Table 1).Over 97% of the isolates were sensitive to chloramphe-nicol, streptomycin and gentamicin. C. coli demon-strated higher resistances (Po0:05) to ampicillin,ciprofloxacin, and nalidixic acid than C. jejuni strains,while C. jejuni were more resistant (Po0:05) tokanamycin than C. coli strains (Table 1). When theisolates were dichotomized across species, C. coli

showed higher (Po0:05) resistance to at least fiveantibiotics than C. jejuni strains (Table 1). Nearly 70%of C. jejuni isolates were resistant to two to fourantibiotics and 25% were resistant to five or moreantibiotics. On the other hand, 40% of C. coli isolateswere resistant to two to four antibiotics and 57% wereresistant to five or more antibiotics. Cluster analysis ofthe antibiograms grouped C. coli and C. jejuni pheno-types in four distinct groups, with 67% of C. coli and64% of C. jejuni assigned to groups’ a2 and A1,respectively (Fig. 1). The antibiogram profile generated13 distinct C. coli and 15 C. jejuni phenotypes with490% similarity and a DI value of 0.552 and 0.742,respectively (Table 2).

3.2. Detection of galE gene

A 497-bp PCR amplicon was observed in �82% oftotal isolates including 91% and 70% of the C. jejuni

and C. coli isolates, respectively (Table 2). The identityof the 497-bp PCR product was confirmed by digestingthe amplicon with HindIII, which cleaved the fragmentinto the expected 430- and 67-bp fragments (data notshown). The specificity of this assay was 100%, whentested against five Escherichia coli, two Salmonella spp.and five Helicobacter spp. ATCC cultures (data notshown).

3.3. SmaI-PFGE analysis

PFGE analysis yielded 6 to 15 DNA fragments foreach genotype ranging from �25 to �500 kb. Thedistribution of Campylobacter genotypes is shown inTable 2. C. coli and C. jejuni dendrogram clusteredrestriction profiles in four and nine distinct groups,respectively (Fig. 2). A total of 15 and 23 restrictionprofiles with 490% similarity was observed among C.

coli and C. jejuni strains, respectively. The calculatedPFGE-DI values for C. coli and C. jejuni were 0.816 and0.941, respectively (Table 2). Based on the size andnumber of DNA fragments, allelic identical clones of C.

coli and C. jejuni were further divided into subgroups C1(n ¼ 9) and J1 (n ¼ 4), J2 (n ¼ 6) and J3 (n ¼ 6),respectively (Fig. 2). To differentiate the clonalityamong these subgroups, strains belonging to each groupwere digested separately with restriction enzyme SalIand the PFGE profile was analysed; the dendrograms

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A1

A2

A3

A4

Fig. 1. UPGMA-dendrograms based on antimicrobial resistance profiles of C. coli (n ¼ 30) and C. jejuni (n ¼ 44) strains. The dendrogram axis

represents percent similarity between isolates. Solid squares represent resistant isolates and blank squares represent susceptible isolates. Antibiotics:

Amp ¼ ampicillin; Cip ¼ ciprofloxacin; Chl ¼ chloramphenicol; Kan ¼ kanamycin; Bac ¼ bacitracin; Str ¼ streptomycin; Sxt ¼ sulfamethoxazo-

le:trimethoprim; Gm ¼ gentamicin; and Na ¼ nalidixic acid. The node selection criteria for clusters a1–a4 and A1–A4 representing C. coli and C.

jejuni, respectively, were based on phenotypes with X80% similarity.

R. Nayak et al. / Food Microbiology 23 (2006) 379–392384

were generated using the same identical cluster selectionmodel as previously described. All nine C. coli clonesfrom group C1 and four C. jejuni clones from group J1remained indistinguishable (490% similarity) fromeach other after digestion with SalI (data not shown).Digestion with SalI split group J2 (n ¼ 6) and J3 (n ¼ 6)into two subclusters with 480% similarity (Fig. 3). C.

coli strains #74–76, 78, 80 and 81 and C. jejuni strains#90–95, 98 and 99 were indistinguishable by bothrestriction enzymes.

3.4. flaA-RFLP analysis

Nearly 88% of Campylobacter species yielded a 1.7-kbfragment after amplification with the flagellin(flaA) gene primers. A total of 26 C. coli and 39 C.

jejuni were successfully genotyped by this methodand flaA groups were assigned on the basis of aphylogenetic dendrogram derived from the bandingpattern analysis (Fig. 4). A total of four C. coli (#24,64, 77 and 97) and five C. jejuni isolates (#53, 68, 71,85 and 93) were untypeable under the conditionsused. The distribution of RFLP-genotypes is shownin Table 2. The RFLP dendrogram generated 17and 27 distinct restriction profiles (490% similarity)in C. coli and C. jejuni strains, respectively. Both C.

coli and C. jejuni dendrograms were clustered intoseven distinct groups (Fig. 4) with DI values of0.895 and 0.942, respectively (Table 2). Only fourgenotypes of C. coli (#75, 76, 79 and 80) were foundto be identical by the SmaI-PFGE and RFLP method(Figs. 2 and 4).

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Table 2

Phenotypic and genotypic dendrogram groups and the prevalence of galE gene in C. coli and C. jejuni strains

Strain no. Species galE Gene Antibiogram pattern SmaI-PFGE pattern RFLP pattern Combined typing

DI ¼ 0.742a DI ¼ 0.941 DI ¼ 0.942 DI ¼ 0.968

25th4 jejuni + A3 P5 R3 C3

27th6 jejuni + A1 P6 ng C1

28th8 jejuni + A2 P1 R7 ng




35th24 jejuni + ng P4 R6 ng

36th25 jejuni + A1 ng ng ng

38th13 jejuni + A1 P9 ng ng

40th29 jejuni + A1 ng R3 ng

42th31 jejuni + Ng ng R6 ng






53th44 jejuni + A2 P5 nt ng

54th43 jejuni + A1 ng ng ng

55tholrhf17 jejuni + A3 ng R3 ng

56tholrR3 jejuni + Ng ng R5 ng

58tho255 jejuni + A4 ng R5 ng

60tho253 jejuni + A3 P4 ng ng

63tho250 jejuni + A1 ng ng ng

65thof7 jejuni � A2 P3 ng C6

66tholrhf9 jejuni � A2 P3 ng C6

67tholr6 jejuni + A2 P9 R4 C4

68tholrhf12 jejuni + A2 P8 nt ng

69tholr4 jejuni + A2 P9 R4 C4

70tholrhf14 jejuni � A2 ng R3 ng

71tholrhf1 jejuni + A3 P8 nt ng

72tholrhf20 jejuni � A2 ng ng ng


85th97 jejuni + A1 P7 nt C2





92th203 jejuni + Ng P5 R1 ng

93th204 jejuni + A3 P2 nt C5






Strain no. Species galE Gene Antibiogram PFGE RFLP Combined typing

DI ¼ 0.552 DI ¼ 0.816 DI ¼ 0.895 DI ¼ 0.91

23th1 coli � a4 p2 ng ng

24th2 coli + a4 ng nt ng

26th5 coli � a3 p3 r7 ng

32th21 coli � a2 p3 r4 g3

33th22 coli + a2 p4 r4 ng

34th23 coli + a2 p3 r4 g3

37-cho coli � a3 ng r7 ng

39th26 coli � a2 p3 ng g4

R. Nayak et al. / Food Microbiology 23 (2006) 379–392 385

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Table 2 (continued )

Strain no. Species galE Gene Antibiogram PFGE RFLP Combined typing

DI ¼ 0.552 DI ¼ 0.816 DI ¼ 0.895 DI ¼ 0.91

44th33 coli + a1 ng r1 ng

49th39 coli + a2 p4 r4 ng

50th40 coli � a2 p3 r4 g4

51th41 coli � a2 ng r5 ng

52th42 coli � Ng ng r6 ng

59tho254 coli + a2 p2 r5 ng

61tho252 coli + a2 p2 r4 ng

64thof3 coli � Ng ng nt ng

73tholrhf22 coli + a3 ng ng ng

74th101 coli + a2 p1 r2 g1

75th102 coli + a2 p1 r2 g1

76th103 coli + a2 p1 r2 g1

77th104 coli + a2 ng nt ng

78th105 coli + a2 p1 r3 g1






87th99 coli + Ng p1 r3 ng

89th200 coli + a2 p1 r6 g2

97th208 coli + a2 p1 nt g2

ng: Non-grouped isolates; nt: non-typeable isolate.aDiscriminatory index (DI) based on Hunter and Gaston (1988) calculations.

Fig. 2. UPGMA-dendrograms based on SmaI-PFGE macrorestriction profiles of C. coli (n ¼ 30) and C. jejuni (n ¼ 44) strains. The dendrogram axis

represents percent similarity between isolates. The DNA fragments are indicated in kilobase (kb) pairs. The node selection criteria for clusters p1–p4

and P1–P9 representing C. coli and C. jejuni, respectively, were based on genotypes with X80% similarity.


ARTICLE IN PRESS

Fig. 3. UPGMA-dendrogram based on isolates in groups J2 and J3 as shown in Fig. 2. The dendrograms on left- and right-hand side represent

digestion with restriction enzymes SmaI and SalI, respectively. The dendrogram axis represents percent similarity between isolates. The DNA

fragments are indicated in kilobase (kb) pairs.

Fig. 4. UPGMA-dendrogram based on flaA-RFLP macrorestriction profiles of C. coli (n ¼ 26) and C. jejuni (n ¼ 39) strains. The dendrogram axis

represents percent similarity between isolates. The DNA fragments are indicated in base pairs (bp). The node selection criteria for clusters r1–r7 and

R1–R7 representing C. coli and C. jejuni, respectively, were based on genotypes with X80% similarity.

R. Nayak et al. / Food Microbiology 23 (2006) 379–392 387

3.5. Combined SmaI-PFGE and antibiogram analysis

C. coli and C. jejuni dendrograms were clustered intofour and six distinct groups, generating 24 and 36different restriction profiles (490% similarity) betweengenotypes, respectively (Fig. 5). The DI values for C.

coli and C. jejuni dendrograms were 0.91 and 0.968,respectively. C. coli strains #75 and #78 and C. jejuni

strains #93 and #94, and #98 and #99 were found to beallelic clones by the combined typing method.

4. Discussion

Due to limited information on turkey productionsscenarios, our discussion has included pertinent broiler

ARTICLE IN PRESS

Fig. 5. Dendrogram based on combined (PFGE+antibiogram) typing methods. The dendrogram axis represents percent similarity between isolates.

Solid squares represent resistant isolates and blank squares represent susceptible isolates. Antibiotics: Amp ¼ ampicillin; Cip ¼ ciprofloxacin;

Chl ¼ chloramphenicol; Kan ¼ kanamycin; Bac ¼ bacitracin; Str ¼ streptomycin; Sxt ¼ sulfamethoxazole:trimethoprim; Gm ¼ gentamicin; and

Na ¼ nalidixic acid. The node selection criteria for clusters c1–c4 and C1–C6 representing C. coli and C. jejuni, respectively, were based on pheno-

and genotype profiles with X80% similarity.


studies that will support the need to address thedynamics of Campylobacter colonization in turkeyproduction environments. The dry conditions of feedand fresh litter are deleterious to Campylobacter species;hence, fresh litter is usually not considered a potentialpreharvest source of infection (Berndtson et al., 1996).However, in our study, 16% of litter samples (mixedwith fecal droppings) were contaminated with Campy-

lobacter, suggesting that wet litter can serve as apotential source of Campylobacter colonization inturkey production facilities. In a recent study of 16broiler flocks from four farms in Arkansas andCalifornia, 14 of the flocks (87.5%) were Campylobac-

ter-positive; only 1% of litter samples tested positive forCampylobacter in Arkansas broiler flocks (Stern et al.,2001). In other surveys, �88–90% of the poultry flocksin the US were colonized with Campylobacter, while inEurope the prevalence varied from 18% to 490%(Stern et al., 2004). The reason for such varied statisticsis unknown. Several factors may contribute to Campy-

lobacter colonization in turkey flocks, including numberof birds per flock, climatic conditions, distance betweenfarms, seasonal variations, geographic locations, proxi-mity to other farm animals, contamination duringbreeding and hatching, and preharvest sources ofcontamination (Newell and Fearnley, 2003). In ourstudy, the turkey litter samples were colonized with

�60% C. jejuni and 40% C. coli strains. The higherincidence of C. jejuni is in agreement with previousstudies in broiler flocks. Oza et al. (2003) tested 387broiler flocks and found that 262 (68%) were colonizedwith Campylobacter spp.; the species composition was91% C. jejuni and 9% C. coli. Elsewhere, 60% of broilerflocks arriving at slaughter facilities were positive forCampylobacter of which 76% were C. jejuni and 24% C.

coli (Avrain et al., 2003).Nearly 13–95% of Campylobacter strains were resis-

tant to ampicillin, aminoglycosides (kanamycin), fluor-oquinolones (ciprofloxacin), nalidixic acid, bacitracinand SXT (Table 1). Nearly 8–20% of the strains wereresistant to two to six antibiotics. The high resistance inCampylobacter strains isolated from poultry farms couldresult from use of antibiotics in veterinary medicine, andthe ability of Campylobacter to acquire resistance genes(Endtz et al., 1991; Iovine and Blaser, 2004). In ourstudy, the turkey feed were mixed with sarafloxacin;however, use of other antibiotics in the sampled turkeyhouses were unknown. The higher frequency of resis-tance could also be because Campylobacter spp. fromturkeys were in contact with the antimicrobials for alonger time; the average grow-out period for turkeys is20–22 weeks compared to 5–6 weeks for broilers. Ourstudy indicates that �48% of C. jejuni and 77% of C.

coli were resistant to ampicillin. Resistance to ampicillin


is usually associated with the ability of Campylobacter toproduce a plasmid-encoded b-lactamase (Lachance etal., 1991), although in some cases these strains weresusceptible to ampicillin even though they produced theenzyme (Saenz et al., 2000). Campylobacter isolates werenot evaluated for b-lactamases in our study. Fluoroqui-nolones have been used in the poultry industry tocontrol mortality in chickens associated with E. coli

infections and to control mortality in turkeys associatedwith E. coli and Pasteurella multocida (Federal Register,2000). In our study, 25% of C. jejuni and 60% of C. coli

were resistant to ciprofloxacin and 39% of C. jejuni and67% of C. coli were resistant to nalidixic acid (Table 1).The emergence of ciprofloxacin-resistant C. coli and C.

jejuni strains may result from the use of sarafloxacin onthe sampled turkey farms. Similar observations werereported by Mc Dermott et al. (2002) in broiler studies.Saenz et al. (2000) observed high frequencies ofresistance (�100%) to nalidixic acid and ciprofloxacinin C. jejuni and C. coli isolated from broiler flocks. Otherstudies have shown that resistance to ciprofloxacin and/or nalidixic acid in broiler flocks ranged from 14% to50% (Hein et al., 2003; Pedersen and Wedderkopp,2003). Fluoroquinolone resistance in Campylobacter

spp. has been attributed to mutations in the gyrA geneand decreased permeability of the bacterial cell wall(Engberg et al., 2001). Additionally, C. jejuni and C. coli

offer intrinsic resistance to fluoroquinolones and b-lactams by an efflux mechanism (Charvalos et al., 1995;Lin et al., 2002; Pumbwe and Piddock, 2002). A multi-drug efflux pump, CmeABC, was identified in C. jejuni,which mediates a two- to fourfold increase in the MICvalues of ampicillin, ciprofloxacin, tetracycline anderythromycin along with ethidium bromide, acridineorange and sodum dodecyl sulfate; the same pump isalso involved in active efflux of ciprofloxacin (Pumbweand Piddock, 2002). The high frequency of resistance totrimethoprim in C. jejuni can be attributed to theacquisition of resistance genes (dfr1 and/or dfr9) codingfor resistant variants of dihydrofolate reductase, thetarget of trimethoprim (Gibreel and Skold, 2000). Thepresence of these genes in C. coli needs to beinvestigated. The low frequency of resistance to chlor-amphenicol, streptomycin and gentamicin in C. coli andC. jejuni (Table 1) suggests that these antibiotics maynot have been used as veterinary antimicrobial agents atthe sampled turkey houses. Campylobacter spp. havealso been found to be susceptible to chloramphenicoland gentamicin in other studies (Saenz et al., 2000; Ozaet al., 2003; Stern et al., 2004).

We screened our isolates for the galE gene as a geneticmarker for Campylobacter infections predisposing in-dividuals with potential risk of post-infection GBS. ThegalE gene was observed in 91% of C. jejuni and 70% ofC. coli isolates, indicating that both Campylobacter spp.may have the potential to cause post-infection compli-

cations. However, more studies will be needed toprovide a better estimate between galE-positive Campy-

lobacter strains on turkey farms to the infection ratesand incidence of GBS in a clinical setting. It must benoted that not all Campylobacter strains, possessing thegalE, can induce GBS because some strains may lackother genes that are necessary to synthesize ganglioside-like structures (B. Fry, pers. comm.). Shi et al. (2002)reported that the wla gene cluster, involved in LPSsynthesis, is highly conserved in Campylobacter strains,and within this cluster both galE and wlaH show highhomology to genes in other Gram-negative organisms.The authors concluded that galE is likely to beconserved among different C. jejuni strains. It is unclearfrom our study why galE gene was not detected in 100%of the isolates. The association between the galE gene inC. coli and the onset of GBS needs further investigation.

Molecular typing methods are essential tools inepidemiological investigation of Campylobacter strains.The genetic instability of the Campylobacter chromo-some could affect the PFGE and RFLP restrictionprofiles (Wassenaar et al., 1998). Other events, such asnaturally occurring multiple recombination events and/or spontaneous genomic rearrangement of mobileelements and lateral gene transfer causing homeoplasisin Campylobacter can also account for differences in therestriction profiles (On, 1998). The PFGE, RFLP andantibiogram profiles discriminated C. coli and C. jejuni

strains into different genotypes, suggesting that thegenetic relatedness among Campylobacter spp. should becompared by more than one typing method (Santeste-ban et al., 1996; Fitzgerald et al., 2001). Nearly 33% ofC. coli strains (#33 and 34; 74–76; 79–80; and 78, 81 and84) and 27% of C. jejuni strains (#29–30; 45–46; 65–66;67 and 69; 93–94; and 98–99) were found to belong to anidentical cluster by all four typing methods, suggestingallelic clonality among these strains (Table 2). On theother hand, C. coli #64 and C. jejuni #48 were mostpheno- and genotypically diverse species in the popula-tion as these strains could not be grouped by any typingmethod (Table 2). The subgrouping of C. jejuni clustersJ2 and J3 by SalI indicates that genetically identicalstrains could be further discriminated by using morethan one restriction enzyme (Fig. 3). A total of 28antibiograms, 38 SmaI-PFGE, 44 RFLP and 60PFGE+antibiogram profiles (490% similarity) weregenerated by combining the C. coli and C. jejuni strains,indicating that a combination of geno- and phenotypingmethod provides greater genetic discrimination betweenCampylobacter species. In general, the restrictionprofiles that were clustered by SmaI-PFGE weregrouped into a different cluster by the RFLP methodand vice versa (Table 2). Nearly 43% and 50% of C. coli

genotypes were found to belong to the same cluster bythe SmaI-PFGE and RFLP method, respectively, while27% and 31% of C. jejuni genotypes were clustered by


the PFGE and RFLP method, respectively, suggestingthat the genetic diversity among C. jejuni populationappears more widespread than C. coli strains on turkeyfarms. When averaged across C. coli genotypes, theRFLP method had the higher discriminatory power(DI ¼ 0.895), followed by SmaI-PFGE (DI ¼ 0.816)and antibiogram profile (DI ¼ 0.552; Table 2), indicat-ing that RFLP is the method of choice in typing C. coli

strains. In other words, if two Campylobacter strains areselected at random from the population, then on �90%of the occasions they would fall into different types bythe RFLP method. However, there may be somedrawbacks of using the RFLP method, which utilizesthe amplification of flaA gene in measuring genetic-relatedness. The flagellin genes in Campylobacter exhibitconcerted evolution (Meinersmann and Hiett, 2000),and recombination between flaA and flaB, a closehomologue, may affect the long-term stability of flaA

in C. jejuni (Harrington et al., 1997). The frequency ofhorizontal transfer of genes for flagellar antigens is alsohigh among C. jejuni strains (Duim et al., 2003). In ourstudy, four C. coli and five C. jejuni strains wereuntypeable (Table 2). The untypeability of flaA can beovercome by performing PCR-RFLP of both genes asdemonstrated by Petersen and Newell (2001). Hence, itis critical to use RFLP as the typing method of choiceonly in Campylobacter strains possessing one or bothflagellar genes. In other cases, the PFGE method can beuseful in addressing the genetic diversity of Campylo-

bacter spp. In case of C. jejuni genotypes, the RFLP-DIvalue (0.942) was not significantly different from theSmaI-PFGE value (DI ¼ 0.941), suggesting eithermethod could be used for typing C. jejuni strains (Table2). The combined SmaI-PFGE and antibiogram typingscheme had the highest DI value, irrespective of thespecies, indicating that a combination of typing methodsshould be employed in discriminating Campylobacter

population on turkey farms.

Acknowledgments

We thank Roger Steele and Don Paine for theirtechnical assistance, and are grateful to Drs. Mark Hart,John Sutherland and Doug Wagner from NCTR fortheir insightful comments and suggestions in preparingthe manuscript. We thank Dr. Hojin Moon from NCTRfor analysing the antimicrobial resistance data. Wethank Dr. Benjamin Fry from the Royal MelbourneInstitute of Technology University, Australia, for hishelpful remarks on the galE section of the manuscript.The primary author appreciates the salary supportreceived from the Oak Ridge Institute of Science andEducation (ORISE) post-graduate research.

Disclaimer: The use of trade names is for identifica-tion purposes only and does not imply endorsement by

the Food and Drug Administration or the US Depart-ment of Health and Human Services. Views presented inthis manuscript do not necessarily reflect those of theFDA.

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