Bacterial strains
39 S. Heidelberg and 5 Escherichia coli isolates included in this study were obtained from human clinical, retail, and agri-food animal sources by CIPARS (Table 1). Antibiotic susceptible S. Heidelberg isolates 1170, 539 and 564 were provided by the Bureau of Microbial Hazards. Phage typing was performed as previously described [19].
Antimicrobial Susceptibility Testing
Resistance to antimicrobials was determined at the Laboratory for Foodborne Zoonoses (LFZ) or the National Microbiology Laboratory (NML) by broth microdilution using the Sensititre™ ARIS Automated Microbiology System (Trek Diagnostic System Ltd, Cleveland Ohio). Breakpoints for resistance were taken from the NCCLS/CLSI guidelines and were as follows: amoxicillin-clavulanic acid; ≥ 32/16 μg/ml, ampicillin; ≥ 32 μg/ml, cefoxitin; ≥ 32 μg/ml, ceftriaxone; ≥ 64 μg/ml, cephalothin; ≥ 32 μg/ml, chloramphenicol; ≥ 32 μg/ml, ciprofloxacin; ≥ 4 μg/ml, gentamicin; ≥ 16 μg/ml, kanamycin; ≥ 64 μg/ml, nalidixic acid; ≥ 32 μg/ml, sulfizoxazole/sulfamethoxazole; ≥ 512 μg/ml, tetracycline; ≥ 16 μg/ml, trimethoprim-sulfamethoxazole; ≥ 4/76 μg/mL [20] and ceftiofur; ≥ 8 μg/ml [21]. The breakpoint used for streptomycin resistance was ≥ 64 μg/ml [22]
Pulsed-field Gel Electrophoresis (PFGE)
PFGE was performed by PulseNet Canada at the NML according to the PulseNet USA protocol using Xba I and Bln I [23]. Pattern analysis and dendrogram construction were performed using the BioNumerics version 4.0 software package (Applied Maths, Austin, TX) with 1% tolerance and 1.5% optimization.
Genomic DNA extractions
Wild type S. Heidelberg or E. coli strains were used to inoculate 4 ml of Luria Bertani [17] broth (Invitrogen, Carlsbad, CA). Alternatively, One Shot® TOP10 Electrocomp™ (Invitrogen) E. coli transformants containing S. Heidelberg blacmy-2 plasmids were inoculated into 6 ml LB broth containing 20 μg/μl cefoxitin (Sigma-Aldrich, Oakville, ON). Cultures were incubated overnight at 37°C with rotation at 200 rpm. Bacterial cells were collected by centrifugation for 5 min at 5000 rpm and resuspended in 2 ml TE buffer (Sigma-Aldrich) (10 mM Tris-HCL, 1 mM EDTA, pH 8.0). Lysozyme (Roche Diagnostics, Indianapolis, IN) (0.5 mg/ml), RNase (Roche Diagnostics) (1.5 μg/ml), and proteinase K (10 mM Tris-HCL pH 7.5, 20 mM CaCl2, 50% glycerol) (Sigma-Aldrich) (0.12 mg/ml) were added to the cell resuspension mixture. Following incubation at 37°C for 1 h, sodium dodecyl sulphate (SDS) (Ambion, Austin, TX) was added to a concentration of 0.1% (wt/vol) and the mixture was incubated at 65°C until clearing occurred. The mixture was then transferred to Phase Lock Light tubes (Eppendorf, Hamburg Germany) for phenol-chloroform DNA extraction using a volume of phenol-chloroform:isoamyl alcohol (25:24:1) (Invitrogen) equal to that of the cell resuspension mixture. Phenol-chloroform:isoamyl alcohol extraction was repeated until the aqueous layer was clear. Following a final extraction with an equal volume of chloroform (Fisher, Ottawa, ON), the aqueous layer was transferred to a new tube. DNA was precipitated at -20°C for 20 min using 0.6 vol of isopropanol (Fisher) and 0.1 vol 3 M sodium acetate (pH 5.5) (Ambion). Following precipitation, DNA was washed with 70% ethanol and resuspended in 200 μl TE buffer. DNA was quantified on a NanoDrop ND-1000 (NanoDrop Technologies, Rockland, DE) and diluted to 20 ng/μl for use as PCR template.
PCR and DNA Sequencing
PCR reactions for tetA, tetB and tetG were performed using 1 unit of Fast start Taq DNA polymerase (Roche Diagnostics) in a reaction mixture containing 1 × Fast start Taq DNA polymerase buffer with MgCl2 (Roche Diagnostics), 1 mM MgCl2 (Roche Diagnostics), 0.2 μM dNTP mixture (Invitrogen), 0.2 μM each primer (Table 2), 20 ng template DNA and distilled water DNAse, RNAase free (Invitrogen) to 25 μl. The thermocycling parameters used for tetA, tetB and tetG reactions containing Fast start Taq DNA polymerase were: initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at a primer specific temperature listed in Table 2 for 30 sec, extension at 72°C for 1 min followed by one final extension at 72°C for 7 min. All other PCR reactions were performed using 1 unit of Platinum Hifi Taq DNA Polymerase High Fidelity (Invitrogen) in a reaction mixture containing 1 × High Fidelity PCR Buffer (600 mM Tris-SO4 [pH 8.9], 180 mM ammonium sulfate) (Invitrogen), 0.2 mM dNTP mixture (Invitrogen), 2 mM MgSO4 (Invitrogen), 0.3 μM each primer (Table 2), 20 ng template DNA and distilled water DNAase, RNAase free (Invitrogen) to 25 μl total volume. The thermocycling parameters used for intA, potE and miaB PCR reactions containing Platinum Hifi Taq were: initial denaturation at 94°C for 5 min followed by thirty cycles of denaturation at 94°C for 30 sec, annealing for 30 sec, at a primer specific temperature listed in Table 2, extension for 60 sec (intA) or 90 sec (potE, miaB) at 68°C followed by one final extension at 68°C for 7 min. The thermocycling parameters used for all other reactions containing Platinum Hifi Taq were: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 sec, annealing for 30 sec, at a primer specific temperature listed in Table 2, extension for 30 sec at 68°C followed by one final extension at 68°C for 7 min. PCR amplicons were resolved by agarose gel electrophoresis on 1.5% agarose gels in 0.5× TBE buffer (Sigma-Aldrich) at 120 V for 60 min. Sequencing was conducted by the DNA Core facility at the NML using an ABI3730 apparatus (Applied Biosystems, Foster City, CA) with the primers used to generate the template (Table 2). Complete sequencing of the blacmy-2PCR product required the design of additional sequencing primers listed in Table 2.
Comparative Genomic Hybridization
DNA microarrays were constructed as previously described using 4492 commercially supplied 70-mer oligonucleotides (Qiagen, Mississauga, ON) representing the coding sequences of the S. Typhimurium LT2 genome as well as all putative open reading frames from Salmonella Genomic Island I (SGI1) [24]. Genomic DNA from test S. Heidelberg and reference S. Typhimuirum LT2 strains was isolated by phenol-chloroform extraction, sheared by nebulization and labelled by Cy3 dCTP or Cy5 dCTP incorporation as previously described [19]. Labelled DNA from test and reference strains was hybridized to the array as previously described with each test-versus-reference comparison performed in triplicate and at least one of the slides hybridized as a dye swap. Following hybridization, slides were sequentially washed in buffer 1 (1 × SSC [3.0 M sodium chloride, 0.3 M sodium citrate] and 0.2 % SDS) for 6 min at 56°C, buffer 2 (0.1 × SSC and 0.2 % SDS) for 4 min at room temperature, and twice in buffer 3 (0.1 × SSC) for 2 min at room temperature. Slides were scanned using an Agilent DNA microarray scanner (Agilent Technologies, Mississauga, ON). Data analysis was conducted as previously described to identify specific loci that were absent or divergent between different S. Heidelberg strains [19].
DNA sequence-based typing (SBT)
SBT was conducted using a previously published scheme based on the fimA, manB, and mdh loci [11]. PCR and sequencing were conducted using primers and annealing temperatures listed in Table 2. Sequence analysis was carried out using Seqman II (DNAstar Inc) and sequences were concatenated to create allelic profiles for each strain.
Plasmid Profiles
A single colony was used to inoculate 8 ml of LB broth and incubated overnight at 37°C with rotation at 200 rpm. Complete plasmid profiles were isolated from 2 ml of overnight culture using a QIAGEN plasmid mini kit (Qiagen) according to manufacturer's directions. Plasmids were resuspended in 25 μl TE buffer (10 μM Tris-HCL pH 8.0, 1 μM EDTA) (Sigma-Aldrich) and resolved by gel electrophoresis on 0.9 % agarose gels in 0.5 × TBE buffer (Sigma-Aldrich) at 90 V for 90 min. Following staining for 20 min in ethidium bromide (2 μg/ml) and destaining for 20 min in ddH2O gels were visualized by UV transillumination using a BioRad GelDoc XR (Bio-rad). Plasmid size was estimated using either a supercoiled DNA ladder (Invitrogen) containing supercoiled plasmids ranging from 2 to 16 kbp.
Electroporation
Transformation of the plasmids encoding blacmy-2 was achieved by adding 5 μl of the plasmid preparations to 50 μl OneShot® TOP10 Electrocomp™ E. coli cells (Invitrogen) in a 0.1 cm chilled cuvette (Cell Projects, Kent, UK). A BioRad Gene Pulser (Bio-Rad) was used to apply a 1.25 kV pulse and 1 ml of S.O.C medium (Invitrogen) was immediately added to the cuvette and the contents transferred to a sterile culture tube. Following incubation of the transformation culture at 37°C for 60 min, 20 and 200 μl aliquots were plated onto LB agar (Invitrogen) plates containing 20 μg/ml cefoxitin (Sigma-Aldrich) and incubated overnight at 37°C. Potential transformant colonies were inoculated into 8 ml LB broth containing 20 μg/ml cefoxitin and incubated overnight at 37°C with agitation. DNA extractions were then conducted and PCR using the blacmy-2 primer set was performed to verify transformants carried the blacmy-2 gene.
Plasmid isolation from transformants
A single transformant colony was inoculated into 8 ml of LB broth with 20 μg/ml cefoxitin and incubated overnight at 37°C with rotation at 200 rpm to create a starter culture. Starter culture was diluted 1/500 into 150 ml LB broth with 20 μg/ml cefoxitin and grown overnight at 37°C with rotation at 200 rpm. Plasmids were isolated from 50 ml of overnight culture using a Qiagen plasmid midi kit according to the manufacturer's directions with the following modification: plasmid DNA was precipitated using 2 ml 7.5 M ammonium acetate (Sigma-Aldrich) in combination with 0.7 vol of isopropanol (Fisher) and resuspended in 100 μl TE buffer.
Restriction Fragment Length Polymorphism (RFLP)
Purified plasmid DNA (25 μl) was digested overnight at 37°C with 20 units of Bgl II (New England Biolabs, Pickering, ON). The resulting plasmid fragments were separated by gel electrophoresis on 0.7 % Tris-acetate-EDTA (TAE) (Gibco) agarose gels at 60 V for 6 h in TAE (400 mM Tris-acetate, 10 mM EDTA). A 1 Kb Plus DNA Ladder (Invitrogen) and Track it λ DNA/Hind III fragments (Invitrogen) were used as molecular size standards. Gels were stained with ethidium bromide (2 μg/ml), destained overnight in ddH2O at 4°C, visualised with UV transillumination and photographed using a Bio-Rad Gel Doc XR. RFLP pattern analysis and was conducted using Bionumerics version 4.0 software with 2% optimization and 10% tolerance. A dendrogram based on RFLP patterns was generated in Bionumerics using the unweighted pair group method of analysis (UPGMA) with a fuzzy band logic coefficient of correlation, 2% optimization, and 10% tolerance.
Southern Blot
RFLP gels were depurinated in 250 mM HCI for 12 min, denatured in a 1.5 M NaCl, 0.5 M NaOH solution for 30 min and finally neutralized in a 1.5 M NaCl, 0.5 M Tris-HCl pH 7.5 solution for 30 min. DNA was transferred by capillary blotting to a positively charged nylon Hybond-N+ (Amersham Biosciences, Little Chalfont, UK) membrane using the TurboBlotter system (Schleicher & Schuell, Keene, NH) according to manufacturer's directions with 10 × SSC (Ambion) transfer buffer. Membranes were rinsed in 6 × SSC (Ambion) and DNA was fixed to the membrane by UV treatment. Nucleic acid labelling and detection was carried out following the manufacturer's directions using the ECL Direct Nucleic Acid Labelling and Detection System (Amersham Life Sciences, Little Chalfont, UK). Labelled probes targeting the aadA1, blacmy-2, dhfRA1, floR, strA, sul1, tetA, and tetB genes were generated from PCR products. PCR amplicons were purified using the QIAquick PCR Purification Kit according to manufacturer's directions, quantified on a NanoDrop ND-1000 and diluted to 10 ng/μl. Amplicons (150 ng) were denatured by boiling for 5 min and snap cooling on ice for 5 min. Probe DNA was labelled at 37°C for 10 min through the addition of equal volumes of DNA labelling reagent (Amersham Life Sciences) and gluteraldehyde (Amersham Life Sciences). Membranes were pre-hybridized at 42°C for 30 min in 25 ml pre-heated ECL gold hybridization buffer (Amersham Life Sciences) containing 0.5 M NaCl and 5 % (w/v) blocking agent (Amersham Life Sciences). Labelled probe was added to hybridization buffer and hybridization was allowed to proceed overnight at 42°C in a Fisher Isotemp hybridization oven (Fisher). Following hybridization, excess probe was removed by washing twice with primary wash buffer (0.5 × SSC, 0.4 % SDS) at 42°C for 20 min and twice with 2 × SSC for 5 min at room temperature. The presence of target gene was detected on Hyperfilm ECL (Amersham) autoradiography film according to manufacturer's directions. Film was developed in a Feline™ developer (Fisher).
Conjugation
Salmonella Heidelberg strains were used as blacmy-2 plasmid donor strains. Recipient E. coli RG192 was serially passaged against rifampicin (Sigma-Aldrich) until resistance to 384 μg/ml was achieved. Cefoxitin (20 μg/ml) and rifampicin (384 μg/ml) were used as selective agents for the donor and recipient strains, respectively. Single colonies of donor and recipient strains from selective plates were inoculated into 8 ml of LB broth containing the appropriate selective antibiotic and grown overnight at 37°C at 200 rpm. Overnight cultures were then sub-cultured into LB broth without selective antibiotic and incubated at 37°C for 5 h at 200 rpm. Recipient and donor cells were combined in a 4:1 ratio in LB broth. Following overnight incubation at 37°C, transconjugants were selected by plating onto LB agar containing cefoxitin (20 μg/ml) and rifampicin (384 μg/ml). The transfer of blacmy-2 was confirmed by performing PCR to detect the presence of the blacmy-2 gene followed by isolation of plasmids from E. coli transconjugants.
