Genetic diversity and virulence gene determinants of antibiotic-resistant Salmonella isolated from preharvest turkey production sources

International Journal of Food Microbiology 91 (2004) 51 – 62 www.elsevier.com/locate/ijfoodmicro

Genetic diversity and virulence gene determinants of antibiotic-resistant Salmonella isolated from preharvest turkey production sources R. Nayak a,*, T. Stewart a, R.-F. Wang a, J. Lin b, C.E. Cerniglia a, P.B. Kenney c a

Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA b Pacific Regional Laboratory SW, US Food and Drug Administration, Los Angeles, CA 90015, USA c Division of Animal and Veterinary Sciences, West Virginia University, Morgantown, WV 26505, USA Received 15 November 2002; received in revised form 1 May 2003; accepted 30 May 2003

Abstract This study evaluated the molecular diversity of 29 Salmonella serotypes isolated from turkey ceca and the production environment. Isolates were resistant to bacitracin (100%), erythromycin (100%), novobiocin (100%), rifampin (100%), streptomycin (62%), gentamicin (52%), spectinomycin (48%), tetracycline (31%), sulfamethoxazole/trimethoprim (SXT) (3%) and tobramycin (3%). The minimum inhibitory concentration (MIC) values ranged from 32 to z 1024 Ag/ml. The pulsed-field gel electrophoresis (PFGE) and ribotyping patterns were identical within each of the serotypes Heidelberg, Worthington and Muenster. The plasmid profiles were identical within each of the Salmonella serotypes. Two different clones of Salmonella anatum were differentiated by PFGE typing but not by ribotyping. Heidelberg isolates from nine turkey ceca and three drinker samples had identical antibiotic resistance, PFGE, ribotype and plasmid patterns, suggesting that transmission of this particular clone may have occurred between the birds and the drinkers. Identical PFGE, ribotype and plasmid patterns were observed in one Salmonella worthington isolate from turkey ceca in one flock and two S. worthington isolates from feeder contents and drinkers from a subsequent flock, suggesting transmission of this pathogen between flocks. Individual and multiple polymerase chain reaction (PCR) analyses revealed the presence of the virulence genes invA, aceK and sopB and the absence of the h-1i gene in all isolates. A combination of genotypic and phenotypic markers can be useful in studying genetic variation among natural salmonellae populations in turkey production and delineating possible transmission pathways. D 2003 Elsevier B.V. All rights reserved. Keywords: Salmonella; Molecular typing; Antibiotic resistance; Turkey

1. Introduction Poultry meat and eggs have been implicated in a large number of salmonellosis cases in humans (Tiet* Corresponding author. Tel.: +1-870-543-7482; fax: +1-870543-7307. E-mail address: [email protected] (R. Nayak). 0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-1605(03)00330-1

jen and Fung, 1995), accounting for nearly one third of deaths among foodborne illnesses in the US (Mead et al., 1999). Salmonella colonizes the intestinal tract of poultry, allowing easy contamination of poultry products during harvest and processing. Once contaminated, meat and animal products that are improperly prepared and/or abused could cause foodborne salmonellosis. The use of antibiotics by the poultry

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industry has been implicated in the emergence and spread of antibiotic-resistant Salmonella strains (van den Boogard and Stobberingh, 1999; Nayak and Kenney, 2003). Reduction of antibiotic-resistant Salmonella in raw and processed poultry necessitates comprehensive control at the breeder farms, hatcheries and production facilities. Bacterial typing methods such as serotyping, antibiotic resistance patterns, plasmid profiling, rapid amplification of polymorphic DNA, restriction fragment length polymorphism and pulsed-field gel electrophoresis (PFGE) have been used to investigate outbreaks and trace Salmonella zoonoses via food to humans (Olsen, 2000; Olsen et al., 1993; Threlfall et al., 1994). Once the route of transmission is identified, control measures can be introduced in an attempt to interrupt the transmission cycle. Without implementation of critical control measures, these ‘‘cycles’’ of Salmonella infection are continuous between humans, animals and the environment. Phenotypic and genotypic characterization of Salmonella serotypes is necessary to delineate possible transmission pathways from hatcheries to processing plants and to determine possible links with clinical strains. Although considerable research has been carried out to understand the molecular diversity of Salmonella ecology in broilers (Olsen, 2000; Olsen et al., 1993; Izumiya et al., 2001; Liebana et al., 2001; Cormican et al., 2002), limited information is available on colonization and the modes of salmonellae transmission in turkeys. The production cycle of turkeys is 20– 22 weeks compared

to 6– 8 weeks in broilers. The longer production cycle may result in different Salmonella ecology in a turkey production facility than in a broiler production facility. This study evaluates the pheno- and genotypic variations among Salmonella serotypes isolated from a turkey production facility.

2. Materials and methods 2.1. Production parameters Four consecutive flocks (F5– F8) were sampled at the West Virginia University turkey production facility from April 1997 to June 1999 (Nayak, 2000). This study was conducted in partnership with the British United Turkeys of America (BUTA), Lewisberg, WV. This facility contained 12 pens on either side of a centrally located service area, for a total of 24 pens. Biosecurity measures were followed as specified by BUTA. Following removal of birds and equipment after each flock, pens were disassembled and litter was removed prior to the placement of the next flock. The production house was cleaned and disinfected, and new litter was added. A detailed description of the flock characteristics is shown in Table 1 (Nayak, 2000). 2.2. Sampling procedures Turkey ceca, litter, drinkers, air, feed samples and environmental swabs were collected from each flock

Table 1 Characteristics of turkey flocks sampled during the survey (adapted from Nayak, 2000) Turkey flocks

BUTA production trials

Sampling period

Sampling frequency (weeks)

Number of pens sampled

Sex of birds

Samplesa

Flock size

Flock 5

6 strains
2 genders

April 1997 to August 1997

2, 10 and 18

12

3 strains
2 feeds

September 1997 to February 1998

6, 13 and 21

6

Flock 7

4 strains
2 feeds

March 1998 to August 1998

5 – 10

Females

Flock 8

4 strains
2 genders

January 1999 to June 1999

0, 2, 10, 14 and 21 2, 15 and 20

6–8

Males and Females

CC, L, D, ES, A and F CC, L, D, ES, A, F and FC CC, L, D, ES, A, F, FC and CC, L, D, ES, A, F and FC

3600b

Flock 6

Males and females Males

1500 2000 3200c

a CC = turkey cecal contents; L = litter samples; D = drinker samples; ES = environmental swabs; A = air samples; F = feed samples; FC = feeder contents. b Combination of 150 females and 150 males per treatment (BUTA production trail). c Combination of 225 females and 175 males per treatment.

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throughout the grow-out period (Table 1). Sample collection and laboratory procedures for isolation and characterization of Salmonella have been described elsewhere (Nayak et al., 2002). Briefly, the turkey ceca were removed, the blind end was snipped with sterile scissors, and cecal contents were emptied into sterile stomacher bags and sealed. Litter samples were collected from the top 5 cm of litter preferably mixed with bird feces and placed in sterile bags and held on ice during transport to the laboratory. Drinkers were swabbed with two or three ply sterile cloth gauzes (25 cm2) held by a pair of sanitized forceps. Environmental swabs were collected from a 16-cm2 area at various locations in the facility using a sterile swab moistened with sterile universal preenrichment (UP) (Difco Laboratories, Detroit, MI, USA) broth. Locations included walls, ventilation fans, feathers and open wounds of sick birds, feathers of dead birds, employee shoes, the feed truck, fans inside the pens, feed storage bins and door handles. Swabs were transferred to 10 ml sterile UP broth. Air samples were collected on 65
15 mm Rodac plates (Fisher Scientific, Pittsburg, PA, USA) containing brain heart infusion (BHI) agar (Difco Laboratories) using a SAS portable high flow air-sampler (Spiral Biotech, Bethesda, MD, USA). The air sampler was set to collect 60 l of air in 20 s. The agar was aseptically transferred to sterile stomacher bags. Feed and feeder samples were collected randomly from each feed shipment by placing a sterile bag in the flow of feed from the auger or from the feed cart. Samples were stored at  4 jC at the production facility prior to transport to the laboratory for analyses. All samples were held on ice during transport to the laboratory and tested for the presence of Salmonella using standard isolation procedures (Nayak and Kenney, 2003). Twenty-nine representative isolates consisting of 21 Salmonella heidelberg, 3 Salmonella anatum, 3 Salmonella worthington and 2 Salmonella muenster were analyzed. 2.3. Antibiotic resistance testing The antibiograms were determined using the diskdiffusion assay (National Committee for Clinical Laboratory Standards [NCCLS], 2000a). Overnight cultures, grown on trypticase soy broth (OD adjusted to 0.5 MacFarland unit), were spread evenly on

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Mueller – Hinton agar (Difco, Becton-Dickinson Microbiological Systems, Sparks, MD, USA). The following antibiotic disks (BD Biosciences, Cockeysville, MD, USA) were tested: ciprofloxacin, 5 Ag; erythromycin, 15 Ag; bacitracin, 10 U; norfloxacin, 10 Ag; novobiocin, 30 Ag; sulfisoxazole, 250 Ag; polymyxin B, 300 U; rifampin, 5 Ag; tetracycline, 30 Ag; streptomycin, 10 Ag; gentamicin, 10 Ag; tobramycin, 10 Ag; ampicillin, 10 Ag; ofloxacin, 5 Ag; chloramphenicol, 30 Ag; kanamycin, 30 Ag; nalidixic acid, 30 Ag; spectinomycin, 100 Ag sulfamethoxazole/trimethoprim (SXT), 1.25/23.75 Ag; ceftriaxone, 30 Ag; cephalothin, 30 Ag; and cefoxitin, 30 Ag. The plates were incubated at 37 jC for 24 h. The zones of inhibition were measured and interpreted as resistant or sensitive according to the manufacturer’s (BD Biosciences) guidelines. 2.4. Minimum inhibitory concentration (MIC) values of test antibiotics Resistant isolates were tested for their MIC values by the macro broth dilution assay (NCCLS, 2000b). Each isolate was suspended in 4 ml of BHI broth and incubated at 37 jC for 18 h. This cell suspension (OD adjusted to 0.5 MacFarland unit) was added to serially diluted BHI tubes containing the appropriate concentration (0.5 – 1024 Ag/ml) of each antibiotic. The tubes were incubated at 37 jC for 24 h and read with indirect light for turbidity (culture growth). 2.5. Restriction analysis of chromosomal DNA Bacterial colonies were suspended in 1 – 2 ml of cell suspension buffer (100 mM Tris – HCl and 100 mM EDTA, pH 8.0). The optical density (610 nm) of the cell suspension was standardized to 1.3 –1.4. An aliquot (400 Al) of cell suspension was mixed with 20 Al of proteinase K (20 mg/ml) and 400 Al of 1% SeaKem Gold agarose (BioWhittaker Molecular Applications, Rockland, ME, USA) and 1% SDS prepared in TE buffer (10 mM Tris – HCl and 1 mM EDTA, pH 8.0). The bacterium –agarose mixture was dispensed into plug molds and allowed to solidify at room temperature for 10– 15 min. Each sample plug was transferred to a 50-ml Nalgene centrifuge tube containing 5 ml cell lysis buffer (50 mM Tris – HCl

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and 50 mM EDTA, pH 8.0 + 1% sodium N-lauroylsarcosinate). Proteinase K was added to each tube to give a final concentration of 0.1 mg/ml. The tubes were incubated in a water bath maintained at 54 jC for 2 h with constant agitation (175 –200 rpm). After proteolysis, plugs in each tube were washed once with 10 – 15 ml of preheated (50 jC) distilled water and four times with preheated (50 jC) sterile TE buffer for 10 – 15 min each time. Each sample plug was digested with 50 U of XbaI (Invitrogen, Carlsbad, CA, USA) at 37 jC for 5 h. The DNA bands were separated on 1% SeaKem Gold agarose by the CHEF-Mapper III PFGE system (Bio-Rad Laboratories, Hercules, CA, USA) using the following electrophoretic conditions: initial switch time, 2.16 s; final switch time, 63.8 s; run time, 18 h; angle, 120j; gradient, 6.0 V/cm; temperature, 14 jC; and ramping factor, linear. After electrophoresis, gels were stained with ethidium bromide and images were captured with the gel documentation system. 2.6. Ribotyping analysis Cells were grown overnight on BHI agar plates at 37 jC and transferred to a lysis buffer. Cells were then incubated at 80jC for 15 min. After air-cooling for 10 min, the heat-inactivated cells were placed in the RiboPrinterR system (Du Pont Qualicon, Wilmington, DE, USA). The RiboPrinterR then performed cell lysis, restriction digest of chromosomal DNA with PvuII (Invitrogen), agarose electrophoresis of restriction fragments, blotting, probing, membrane processing and image recording. RiboPrinterR analytical software normalized the number, position and

relative intensities of rRNA operon specific DNA fragments and archived a digital record of each isolate. Isolates were characterized by clustering their RiboPrintR within a specific similarity range to form RiboGroups. 2.7. Plasmid analysis Bacterial cells were grown overnight at 37 jC in 2 –5 ml of Luria – Bertani medium. Cells were harvested and lysed, and plasmid DNA was extracted using the plasmid isolation kit (Qiagen, Valencia, CA, USA). Plasmid DNA was electrophoresed in 0.8% agarose gels with 1
Tris – borate – EDTA (TBE) buffer. Supercoiled DNA ladder (Invitrogen) was used as a molecular weight marker. 2.8. Polymerase chain reaction (PCR) detection of virulence genes Freshly grown cells (18 – 24 h) on BHI plates were suspended in 0.5 ml of sterile deionized water and heated in a boiling water bath for 20 min. The samples were cooled immediately in an ice bath for 5 min and centrifuged. The supernatants were used as DNA templates for PCR. Individual and multiplex PCR was used to screen each Salmonella isolate for the presence of virulence genes (h-1i and sopB) using specific primers (Way et al., 1993; Mirold et al., 2001) as shown in Table 2. The primers for invA and aceK were designed with the DNASTAR primer selection software (DNASTAR, Madison, WI, USA) using published sequences from the GenBank. For the amplification of individual genes, the PCR reaction

Table 2 PCR primers used for amplification of virulence genes in Salmonella isolates Target gene

Oligonucleotide sequence

Length (bp)

Position and orientation on DNA sequencea

invA

F-5VTAT CGC CAC GTT CGG GCA A-3V R-5VTCG CAC CGT CAA AGG AAC C-3V F-5VAGC CTC GGC TAC TGG TCT TG-3V R-5VCCG CAG CAA GAG TCA CCT CA-3V F-5VCAG GGC GAG GGT ATG AAA CAC-3V R-5VAAC TGC GAT TCT TCG GTA GAA C-3V F-5VATG CAA ATA CAG AGC TTC TAT CA-3V R-5VGGC ATA AAG GGA CAG CAC A-3V

19 19 20 20 21 22 23 19

276 ! 294 532 p 550 624 ! 643 796 p 777 19 ! 39 209 p 188 5055 ! 5077 6881 p 6899

h-1i aceK sopB a

Amplification region (bp) 275 173 191 1700

The nucleotide position is determined from the initiation codon of the gene. The arrows indicate orientation of the oligonucleotide.

R. Nayak et al. / International Journal of Food Microbiology 91 (2004) 51–62

mixture contained 2.5 Al of DNA template, 12.5 Al of 2
Qiagen Taq PCR Master mix [Tris –HCl, KCl and (NH4)2SO4 buffer, pH 8.7; 3 mM MgCl2, 400 AM of each dNTP and 0.05 U/Al of Taq polymerase], 0.5 Al of each primer mix (25 AM stock concentration) and deionized water to a final volume of 25 Al. The reaction mixture was amplified in a 9700 GeneAmpR PCR system (Applied Biosystems, Foster City, CA, USA) using the following conditions: heat denaturation at 95 jC for 1 min, 35 cycles with denaturation at 95 jC for 20 s, annealing at 55 jC for 20 s and extension at 72 jC for 2 min, and final DNA extension at 72 jC for 4 min. For multiplex PCR, the final primer concentrations in the reaction mixture were 0.1 AM of invA primer mix, 0.1 AM of aceK primer mix and 2 AM of sopB primer mix. A positive control of Salmonella typhimurium ATCC 14028 containing all four genes was included in each run.

3. Results and discussion 3.1. Antibiotic resistance and MIC values Salmonella was isolated from turkey ceca, litter, drinkers, environmental swabs, feed and feeder contents of a turkey production facility. Isolates were resistant to bacitracin (100%), erythromycin (100%), novobiocin (100%), rifampin (100%), streptomycin (62%), gentamicin (52%), spectinomycin (48%), tetracycline (31%), SXT (3%) and tobramycin (3%), and sensitive to ampicillin, ofloxacin, chloramphenicol, kanamycin, nalidixic acid, ciprofloxacin, norfloxacin, polymyxin-B, ceftriaxone, cephalothin and cefoxitin (Table 3). The high resistance was further reflected in the MIC values for tetracycline (32 – 256 Ag/ml), spectinomycin (z 512 Ag/ml), streptomycin (z 256 Ag/ml), gentamicin (z 256 Ag/ml), erythromycin (32 to >512 Ag/ml), rifampin (512 – 1024 Ag/ml) and novobiocin (>1024 Ag/ml). The selective pressure resulting from widespread subtherapeutic use of antimicrobials by the food animal industry and hospitals may be the primary force in the development of resistance (Tollefson and Miller, 2000). The practice of dipping hatching turkey eggs in gentamicin sulfate solutions by turkey breeders to prevent mycoplasmosis (Hirsch et al., 1983) could explain the large number of gentamicin-resistant strains. Additionally,

55

gentamicin, spectinomycin and norfloxacin have been injected in day-old poults to prevent Escherichia coli infections (Poppe et al., 1995). Antibiotics such as sulfonamides, tetracyclines, neomycin, spectinomycin and h-lactamases used in the treatment of poults and turkeys infected with Salmonella arizonae, in acute outbreaks of paratyphoid or other infections and prevention of such infections in turkeys, any of which may account for the drug resistances observed in the present study. Resistance of Salmonella to gentamicin in this study was higher than the percentage of gentamicin-resistant Salmonella (25.8%) from turkey flocks in Canada (Poppe et al., 1995). Furthermore, Salmonella resistance to spectinomycin and tetracycline in our study was lower than the percentage of spectinomycin (97.6%) and tetracycline-resistant (38.1%) Salmonella reported by Poppe et al. (1995). However, in both studies, Salmonella was found to be sensitive to quinolones such as ciprofloxacin, nalidixic acid and norfloxacin. This observation is of importance to public health because ciprofloxacin is increasingly used in the treatment of salmonellosis in humans (Angulo et al., 2000). Although treatment of salmonellosis using ciprofloxacin has met with considerable success (Asperilla et al., 1990), fluoroquinolone-resistant Salmonella have been reported in humans and poultry (Threlfall et al., 1997; Angulo et al., 2000; Helmuth, 2000). Several strains of Salmonella, resistant to multiple antibiotics, have been isolated from farm animals and foods of animal origin (Nayak, 2000; Tollefson and Miller, 2000). In this study, Salmonella isolates were resistant to multiple antibiotics. S. anatum and S. worthington had identical antibiotic resistance phenotypes, while S. heidelberg and S. muenster showed a different antibiotic resistance profile (Table 3). All isolates showed a common resistance to erythromycin, bacitracin, novobiocin and rifampin. Multiple drug resistance in Salmonella may result from random chromosomal mutations and transfer of resistance genes via conjugation and transformation of the resistance transfer factor (RTF) and resistance (R) determinants (Salyers and Whitt, 1994; Helmuth, 2000). 3.2. PFGE, riboprinting and plasmid analysis Molecular typing methods have been used to study the genetic diversity of antibiotic-resistant Salmonella

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Table 3 Antibiotic resistance profiles of Salmonella isolatesa and the MIC values ID no.

5F28 5F29 5F31 5F103 5F105 5F110 5F111 5F112 5F114 5F116 5F118 5F119 5F128 5F129 5F130 5F131 5F133 5F138 5F139 5F140 5F148 5F149 5F150 5F155 5F156 6F14 6F15 8F3 8F7 14028

Salmonella serotypes

Source

S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.

Drinker Drinker Drinker Swab Swab Drinker Drinker Drinker Drinker Drinker Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Feeder contents Litter Litter Swab ATCC

anatum anatum anatum heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg worthington heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg worthington worthington muenster muenster typhimurium

Antibiotic resistance phenotypes

MIC (Ag/ml) Te

St

Ge

Spt

Er

Ri

No

Te, St, Er, B, No, Ri Te, St, Er, B, No, Ri Te, St, Er, B, No, Ri Er, B, No, Ri Te, Sxt, Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri Te, St, Spt, Ge, Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri Te, Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri St, Spt, Ge, Er, B, No, Ri Er, B, No, Ri Te, St, Spt, Ge, Er, B, No, Ri Er, B, No, Ri Te, Er, B, No, Ri Te, Er, B, No, Ri Er, B, No, Ri St, Ge, To, Er, B, No, Ri Er, B, No, Ri

512 256 256 – 128 – – – – 256 – – – – – – 32 – – – – – – 256 – 32 32 – – –

256 256 256 – – >256 >256 – >256 >256 >256 – >256 >256 >256 >256 – >256 – >256 >256 >256 – >256 – – – – >256 –

>256 >256 >256 – – 256 256 – 256 >256 256 – 256 256 >256 256 – 256 – 256 >256 256 – 128 – – – – >256 –

– – – – – >512 >512 – >512 >512 >512 – >512 >512 >512 >512 – >512 – >512 >512 >512 – >512 – – – – – –

128 128 256 512 128 >512 128 512 512 512 512 256 512 512 512 256 128 128 256 512 128 128 256 256 256 64 128 512 32 128

512 512 256 128 256 512 1024 512 1024 1024 1024 1024 1024 1024 512 512 512 1024 1024 512 512 1024 1024 1024 512 1024 1024 1024 1024 1024

>1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024 >1024

Abbreviations: Te = tetracycline; St = streptomycin; Ge = gentamicin; Spt = spectinomycin; Er = erythromycin; To = tobramycin; Ri = rifampin; No = novobiocin; and B = bacitracin. a Each isolate was sensitive to ciprofloxacin, norfloxacin, polymyxin B, ofloxacin, nalidixic acid, ampicillin, chloramphenicol kanamycin, ceftriaxone, cephalothin and cefoxitin.

(Olsen et al., 1993; Threlfall et al., 1994). In this study, PFGE, ribotyping and plasmid profiling methods were used to determine the degree of genomic polymorphism. The PFGE analysis revealed five distinct patterns (P1 – P5) ranging from 11 to 20 DNA fragments (Table 4). Analysis of S. heidelberg and S. anatum revealed 13 distinct DNA fragments ranging in size from f 23 to 679 kb and from f 35 to 873 kb, respectively (Fig. 1). The PFGE patterns of S. worthington and S. muenster showed 14 (f 35 to 873 kb) and 10 (f 145 to 388 kb) DNA fragments, respectively. An identical PFGE pattern was observed in S. heidelberg, S. worthington and S. muenster isolates (Fig. 1). Two S. anatum genotypes were

isolated from drinker samples. The genetic homogeneity among Salmonella isolates was also characterized by the ribotyping method. S. anatum and S. heidelberg ribotype patterns revealed eight fragments ranging from 2 to 9.5 kb and from 2 to 15 kb, respectively, while six fragments were observed in S. worthington and S. muenster isolates ranging from 2 to 9 kb and from 2 to 13 kb, respectively. Ribotyping analysis revealed a genomic cluster unique to each of the four Salmonella serotypes (Fig. 2). However, the ribotyping pattern was different between Salmonella serotypes. To further confirm the genetic homogeneity among the 21 S. heidelberg isolates from turkey ceca, drinkers and environmental swabs, chro-

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Table 4 Pulsed-field gel electrophoresis, plasmid profile and virulence gene analysis of Salmonella isolates PFGE pattern

ID no.

P1 P1 P2 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P3 P4 P4 P4 P5 P5 –

5F28 5F31 5F29 5F103 5F105 5F110 5F111 5F112 5F114 5F116 5F118 5F119 5F128 5F129 5F130 5F131 5F138 5F139 5F140 5F148 5F149 5F150 5F155 5F156 5F133 6F14 6F15 8F3 8F7 14,028

Salmonella species

S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.

anatum anatum anatum heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg heidelberg worthington worthington worthington muenster muenster typhimurium

Source

Drinker Drinker Drinker Swab Swab Drinker Drinker Drinker Drinker Drinker Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Turkey ceca Feeder contents Litter Litter Swab ATCC

mosomal DNA from these isolates was digested with PstI prior to ribotyping. Analysis with PstI also revealed an identical ribotype cluster consisting of 6 DNA fragments ranging from 9 to 50 kb among the 21 S. heidelberg species (data not shown). There is limited information available on molecular typing and characterization of Salmonella isolated from turkey production. Molecular typing methods such as PFGE, ribotyping and plasmid profiling, in combination with phenotypic methods such as serotyping and antibiogram patterns, used in this study shed some light on the possible transmission of antibiotic-resistant Salmonella within a turkey production facility. The PFGE patterns of S. heidelberg, S. worthington and S. muenster were identical, suggesting that these isolates originated from the same clone (Fig. 1). S. anatum isolates from the drinkers

Plasmid (kb)

3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 1, 1, 1, 1, 1,

3.5, 4, 8, 9, 10 3.5, 4, 8, 9, 10 3.5, 4, 8, 9, 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 9, 10 9, 10 9, 10 2.5, 3, 5.5, 6, 7 2.5, 3, 5.5, 6, 7 –

Virulence genes invA

aceK

sopB

h-1i

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

                             +

(5F28 and 5F29), with identical antibiotic resistance phenotypes (Table 2), showed two different PFGE patterns (P1 and P2; Table 3). Although two different PFGE patterns were observed for S. anatum (Fig. 1), the ribotyping pattern for S. anatum was identical (Fig. 2), suggesting the superior discriminatory power of the PFGE technique in this case. S. heidelberg isolates from nine turkey ceca (ID nos. 5F118, 128, 129, 130, 131, 138, 140, 148 and 149) and three drinkers (ID nos. 5F110, 111 and 114) had identical antibiotic resistance profiles (streptomycin, spectinomycin, gentamicin, erythromycin, bacitracin, novobiocin and rifampin; Table 2), PFGE patterns (P3; Table 3) and ribotyping clusters (Fig. 2), and transmission of S. heidelberg may have occurred between the bird and the drinkers. Identical clones of S. worthington (ID nos. 5F133, 6F14 and 6F15) with similar antibiotic

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Fig. 1. Pulsed-field gel electrophoresis patterns of Salmonella with restriction enzyme XbaI. Lanes 1 – 3, S. anatum; lanes 4 – 24, S. heidelberg; lanes 25 – 27, S. worthington; and lanes 28 – 29, S. muenster. P1 – P5 represents the PFGE patterns. Lane M = low range molecular weight markers; lane C = control strain of S. typhimurium ATCC 14028.

resistance phenotypes (tetracycline, erythromycin, bacitracin, novobiocin and rifampin), PFGE clusters (P4) and ribotype patterns were found in two subsequent flocks, designated with ID nos. 5F and 6F (Table 4), suggesting the possible transmission of S. worthington from flock 5 to flock 6 (Table 1). Plasmid analysis has been successfully used as an epidemiological tool to investigate salmonellosis, discriminate Salmonella strains and trace antibiotic resistance transfer to transconjugants (Poppe et al., 1993; Millemann et al., 1995; Rychlik et al., 2000). The plasmid profile in this study was identical within each Salmonella serotype (Table 4). S. anatum isolates from drinker samples, with plasmid sizes of approximately 3, 3.5, 4, 8, 9 and 10 kb, were associated with identical antibiotic resistance profiles (tetracycline, streptomycin, erythromycin, bacitracin, novobiocin and rifampin) but two distinct PFGE patterns (P1 and P2). The 3- and 10-kb plasmids detected in all

S. heidelberg isolates exhibited resistance to a wide range of antibiotics such as tetracycline, streptomycin, erythromycin, bacitracin, novobiocin, rifampin, streptomycin, spectinomycin, gentamicin and SXT (Table 3). Three identical plasmids (1, 9 and 10 kb) were detected in S. worthington isolates that were resistant to tetracycline, erythromycin, bacitracin, novobiocin and rifampin, while a distinct plasmid profile (1, 2.5, 3, 5.5, 6 and 7 kb) was found in S. muenster isolates that were resistant to streptomycin, gentamicin, tobramycin, erythromycin, bacitracin, novobiocin and rifampin (Table 3). Plasmid encoded resistance to cefotaxime and ampicillin has been reported in S. typhimurium and S. enteritidis, respectively (Vatopoulos et al., 1994; Gazouli et al., 1998). Although plasmids of high molecular weight, such as 36- and 60-MDa plasmids in S. enteritidis and S. typhimurium, respectively, are attributed to virulence and antibiotic resistance (Poppe et al., 1993; Rychlik et al., 2000),

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Fig. 2. Ribotyping profiles of Salmonella strains isolated from a turkey production facility.

low-molecular-weight plasmids in Salmonella contribute to differences in phage typing results (Gado et al., 1998). 3.3. PCR analysis of virulent genes Several Salmonella-specific virulence genes that are involved in pathogenicity have been identified (Ba¨umler et al., 2000). Taking advantage of these Salmonella-specific genes, oligonucleotide probes have been designed to develop rapid and reliable PCR-based assays to detect salmonellae from food, feed and environmental sources (Ferretti et al., 2001; Gentry-Weeks et al., 2002). In this study, a multiplex PCR assay was developed to screen Salmonella iso-

lates for the virulence genes invA, aceK, sopB and h1i (Fig. 3). The invA (275 bp) was designed from nucleotide sequence deposited in the GenBank under the accession number U43237. PCR analysis revealed the presence of invA in all isolates (Table 3). The use of genes in the inv family (invA, B, C and D) as a potential target for Salmonella identification has been suggested since these genes were shown to be present in a number of Salmonella strains (Galan and Curtiss, 1991; Stone et al., 1994; Swamy et al., 1996). The invA is the first gene in an operon that triggers the internalization of S. typhimurium in cultured epithelial cells (Galan and Curtiss, 1989). The use of this gene as a suitable PCR target with potential diagnostic applications for specific detection of Salmonella from

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Fig. 3. Agarose gel electrophoresis of amplicons generated by individual (lanes 1 – 4) and multiplex (lanes 5 – 9) PCR amplification of Salmonella using gene specific primers described in Table 1. Lane 1, 275-bp invA amplicon; lane 2, 191-bp aceK amplicon; lane 3, 173-bp h-1i amplicon; lane 4, 1.7-kb sopB amplicon; and lanes 5 – 9, multiplex PCR products of S. anatum (5F28), S. heidelberg (5F103), S. heidelberg (5F119), S. worthington (6F14) and S. muenster (8F3), respectively. Lane M contains molecular weight standards (100-bp DNA ladder).

human and animal origin was demonstrated by Rahn et al. (1992). The authors showed that the invA was detected in 99.4% of Salmonella strains and could not be amplified from non-Salmonella strains, suggesting that the invA gene contains sequences unique to Salmonella. The aceK gene encodes a bifunctional regulatory enzyme (IDH K/P) that catalyzes phosphorylation and dephosphorylation of isocitrate dehydrogenase (IDH) and thereby controls the flux of isocitrate through the tricarboxylic acid cycle and the glyoxylate bypass (Nelson et al., 1997). The primers from detecting aceK gene were designed from nucleotide sequence deposited in the GenBank under the accession number U43359. The PCR assay could detect the aceK gene with a PCR product of 191 bp in all isolates (Table 3). The sopB gene is a translocated effector protein of the SPI Type III system that facilitates induction of apoptosis in macrophages, interleukin production, membrane ruffling and invasion into nonphagocytic host cells (Mirold et al., 2001). This gene (1.7 kb) was also detected in all Salmonella isolates (Table 3). The presence of invA, aceK and sopB genes in all Salmonella isolates could be potentially used as diagnostic applications for specific detection of Salmonella from human and animal origin. However, the specificity of these genes should be further confirmed individually

and in a multiplex PCR-based assay using non-Salmonella strains. The h-1i flagellin genes have been successfully used in conjunction with the phoP and hin genes for species-specific detection of motile Salmonella DNA (Way et al., 1993). The h-1i gene is involved in the control of phase variation and motility of Salmonella species. The PCR assay in this study could not detect the h-1i gene with an amplicon size of 173 bp in all isolates (Table 3). The absence of h-1i in S. anatum, S. heidelberg, S. worthington or S. muenster suggests either the absence of H-antigen or the number of copies of the H-antigen are too low to be amplified in these species. The flagellated H-component antigenic properties of Salmonella were not tested in this study. The positive control (S. typhimurium ATCC 14028) used in this study, possessing the H-antigen (Way et al., 1993), tested positive for the h-1i gene (Fig. 3). Overall, the use of multiplex PCR analysis allowed specific and simultaneous detection of virulence genes in Salmonella. The ubiquitous nature of Salmonella in the environment creates a significant challenge for animal agriculture to produce meat animals free of foodborne pathogens. Molecular typing can be used as a valuable epidemiological tool in elucidating possible movement and transmission pathways of Salmonella within

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turkey production facilities and from breeder flocks and hatcheries to the consumers. Because the number of Salmonella isolates used in this study was limited, a comprehensive collection of isolates with detailed information on samples collected, sample locations, sampling frequencies and sampling periods from hatcheries, production environment and processing plants is warranted to identify the source and possible modes of transmission. A knowledge of pre- and postharvest sources responsible for Salmonella colonization and the pheno- and genotypic characterization of isolates may help integrators and turkey producers: (1) define critical, pre- and postharvest control points on the farm and processing units because intervention at these points may reduce and/ or eliminate horizontal transmission and Salmonellapositive birds arriving at the processing plants; and (2) design hazard analysis and critical control point (HACCP) protocols to safeguard fresh turkey against Salmonella contamination during processing and deliver Salmonella-free turkeys to consumers. More importantly, this knowledge would facilitate Salmonella control by means other than subtherapeutic use of antibiotics.

Acknowledgements The work was supported in part by the postgraduate research program at the NCTR and administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department for Science and Education and the US Food and Drug Administration. We thank the NCTR staff members for their useful comments and suggestions in preparing the manuscript.

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