The material for the study consisted of field isolates of Campylobacter spp. obtained from the gut (cecum) of 140 broiler chickens directly after slaughter in slaughterhouses in southeastern Poland in September and October. The birds were from different indoor flocks. Presumptive identification of Campylobacter spp. isolates was based on colony morphology, Gram staining, and growth in microaerobic conditions. Initial isolation was carried out in Bolton Broth (Oxoid Ltd., UK). The cultures were incubated at 37°C for 48 hr in microaerophilic conditions (5% O2, 10% CO2, 85% N) in the CampyGen system (Oxoid Ltd.). On media that showed growth of gray, flat, and moist bacterial colonies with a tendency to expand, single colonies belonging morphologically to the Campylobacter spp. type were collected, directly transferred to selective mCCDA agar, and incubated at 41.5°C for 48 hr in microaerophilic conditions (Dudzic et al., 2016 ). The isolates were stored at −80°C in the Microbank system for storage of micro‐organisms (Biocorp, PL).
Campygen system
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The CampyGen system is a laboratory instrument designed to create a microaerophilic environment suitable for the growth of Campylobacter species. It generates a controlled gas mixture of nitrogen, hydrogen, and carbon dioxide to support the growth of these fastidious organisms.
Automatically generated - may contain errors
Lab products found in correlation
Vancomycin, by Merck Group (3 mentions)
Vancomycin, by Thermo Fisher Scientific (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Gc agar, by Thermo Fisher Scientific (2 mentions)
Bolton broth, by Thermo Fisher Scientific (1 mentions)
Microbank system, by Biocorp (1 mentions)
Brucella broth, by BD (1 mentions)
Mrs agar, by Thermo Fisher Scientific (1 mentions)
Atcc 43504, by American Type Culture Collection (1 mentions)
Nctc11168, by American Type Culture Collection (1 mentions)
Trimethoprim, by Merck Group (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Amoxicillin, by Thermo Fisher Scientific (1 mentions)
Tetracycline, by Thermo Fisher Scientific (1 mentions)
Campylobacter growth supplement, by Thermo Fisher Scientific (1 mentions)
Campylobacter blood free selective agar base, by Thermo Fisher Scientific (1 mentions)
11 protocols using campygen system
1
Isolation and Identification of Campylobacter from Broilers
2
Culturing and Storing Campylobacter jejuni
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The C. jejuni strains used in this study are listed in Table 1 . C. jejuni was stored at -80°C in Brucella broth containing 10% glycerol. C. jejuni was routinely grown on TSA-blood plates (1.5% pancreatic digest of casein, papaic digest of soybean, 0.5% sodium chloride, 1.5% agar and 5% defibrinated sheep blood) for 2 days at 42°C under a microaerophilic atmosphere generated with the CampyGen system (Oxoid). For liquid culture, C. jejuni was grown in Brucella broth (BD Biosciences) containing 10 g/L pancreatic digest of casein, 10 g/L peptic digest of animal tissue, 1 g/L dextrose, 2 g/L yeast extract, 5 g/L sodium chloride, 0.1 g/L sodium bisulfite.
3
Cultivation and Characterization of Helicobacter pylori
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Helicobacter pylori (HP) type strain ATCC 43504 (or NCTC 11637), used in this study, was obtained from American Type Culture Collection (USA). The identity of the pathogen was confirmed by PCR assay of H. pylori specific gene glmM [19 ]. HP was grown in 10 ml of liquid brain heart infusion medium (BHI; Oxoid, UK) supplemented with 10% (w/v) fetal bovine serum (FBS; Oxoid, UK), and incubated under microaerophilic conditions generated by the CampyGen system (Oxoid) at 37°C, as described [20 (link)]. For in vitro and in vivo studies, bacteria were harvested in exponential phase (OD600 nm; 0-6-0.8) by centrifugation (3500 g for 5 min), and suspended in Mueller Hinton broth (MH, Oxoid, UK) for antimicrobial activity or in sterile NaCl 0,9% to infect mice respectively. The probiotic Lactobacillus paracasei was obtained from by the Department of Microbiology of the University of Naples Federico II. Cell free supernatant (CFS) of Lactobacillus paracasei was obtained as described previously [21 –22 (link)]. The supernatant was tested to identify the presence of bacteria by plating serial dilutions on MRS Agar (Oxoid England). No bacteria were identified after filtration [21 –22 (link)].
4
H. pylori Infection Assay in MKN-28 Cells
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H. pylori strain ATCC 43504 was grown in 10 ml of liquid brain heart infusion medium (BHI; Oxoid, UK) supplemented with 10% foetal bovine serum (FBS; Oxoid), and incubated under microaerophilic condition generated by the CampyGen system (Oxoid) at 37 °C78 (link). Bacteria were harvested at mid exponential phase, centrifuged (4.7 × 103 g; 5 min), filtered (0.22 µ) and added (140 µl/well; 30 min and 1 h) to growing MKN-28 cells.
5
Isolation and Characterization of Campylobacter jejuni Strains
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Campylobacter jejuni reference strains RM1221 and NCTC11168 (ATCC 700819) were acquired from Cedarlane (Ontario, Canada). The other strains were isolated from chicken at the time of slaughter in slaughterhouse located in Quebec, Canada, as part of a previously published study (Thibodeau et al. 2013 (link), Table1 ). C. jejuni strains were routinely grown on tryptic soy agar (TSA) supplemented with 5 % defibrinated sheep blood (TSA-blood). The plates were incubated at 42 °C in a microaerophillic atmosphere generated with the CampyGen system (Oxoid).
Campylobacter jejuni reference strains RM1221 and NCTC11168 (ATCC 700819) were acquired from Cedarlane (Ontario, Canada). The other strains were isolated from chicken at the time of slaughter in slaughterhouse located in Quebec, Canada, as part of a previously published study (Thibodeau et al. 2013 (link), Table
Campylobacter jejuni strains used in this study
| Name | Origin | Condition of isolation | Reference |
|---|---|---|---|
| NCTC11168 | Human | Clinical isolate | Ahmed et al. (2002 (link)) |
| RM1221 | Chicken | Store-bought chicken carcass | Miller et al. (2000 (link)) |
| G2008b | Chicken | Caecal content at time of slaughter | Thibodeau et al. (2015 (link)) |
| L2003a | Chicken | Caecal content at time of slaughter | Thibodeau et al. (2015 (link)) |
| D2008a | Chicken | Caecal content at time of slaughter | Thibodeau et al. (2015 (link)) |
| F2008a | Chicken | Caecal content at time of slaughter | Thibodeau et al. (2015 (link)) |
| F2008d | Chicken | Caecal content at time of slaughter | Thibodeau et al. (2015 (link)) |
| A2008a | Chicken | Caecal content at time of slaughter | Thibodeau et al. (2015 (link)) |
| T2003a | Chicken | Caecal content at time of slaughter | Thibodeau et al. (2015 (link)) |
6
Characterization of C. jejuni Mutants
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C. jejuni strain 81–176, a highly invasive isolate from a raw milk outbreak [31] (link), was used as the WT parental strain. All other strains are listed inTable 1
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C. jejuni strain 81–176, a highly invasive isolate from a raw milk outbreak [31] (link), was used as the WT parental strain. All other strains are listed in
7
Cultivation and Manipulation of H. pylori
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Construction of the various isogenic mutants of H. pylori strain P12 has been described in detail previously (Gorrell et al., 2013 (link)). H. pylori strain 7.13 and its isogenic ΔcagA mutant have also been described elsewhere (Franco et al., 2005 (link)). H. pylori strains were routinely cultured on GC agar (Oxoid) supplemented with 10% (v/v) horse serum (Invitrogen), vitamin mix, vancomycin, and nystatin as described previously (Kwok et al., 2002 (link)). For infection experiments, GC agar-cultured H. pylori was used to inoculate Heart Infusion (HI) broth (Oxoid) supplemented with 10% (v/v) FBS, 1% (v/v) vitamin mix and 10 μg/ml vancomycin (Sigma-Aldrich). Kanamycin sulfate (15 μg/ml) or chloramphenicol (4 μg/ml) was added as required for the culture of H. pylori mutant strains. All H. pylori cultures were grown at 37°C under microaerophilic conditions (CampyGen™ system; Oxoid); liquid cultures were incubated with gentle agitation at 120 rpm to an O.D550nm of 0.8–1.2 prior to use in infections.
8
Mammalian Cell and Helicobacter pylori Culture
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Mammalian cell and H. pylori culture was performed as described previously [12 (link)]. Gastric adenocarcinoma epithelial cell line AGS [13 (link)] was routinely cultured in RPMI (Invitrogen Corp, Carlsbad, CA, USA) supplemented with 10% (v/v) foetal bovine serum (FBS, Invitrogen) in a humidified incubator at 37°C with 5% CO2. H. pylori strains (S1 Table ) were routinely cultured on GC agar (Oxoid, Basingstoke, UK) supplemented with 10% (v/v) horse serum (Invitrogen), vitamin mix, vancomycin and nystatin as described previously [14 (link)]. For cell-culture inoculum, H. pylori strains were cultured in brain heart infusion (BHI) or heart infusion (HI) broth (Oxoid) supplemented with 10% (v/v) FBS, vitamin mix, and vancomycin (Sigma, St Louis, MO, USA). H. pylori growth medium was further supplemented with chloramphenicol (Cm; 8 μg/ml for transformant selection, 4 μg/ml for routine culture) or kanamycin sulphate (Km;15 μg/ml) as required. All H. pylori culture was performed at 37°C under microaerobic conditions generated using the CampyGen system (Oxoid); broth cultures were shaken at 120 rpm. E. coli strain DH5α was propagated as described previously [15 (link)].
9
Antibiotic Susceptibility of H. pylori
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Eight antimicrobial agents were tested against each H. pylori isolate using the antibiotic test discs (amoxicillin, levofloxacin clarithromycin and tetracycline) (Oxoid Ltd). Other drugs are metronidazole, ciprofloxacin, cefuroxime and tinidazole. Inoculum from the subculture was made and streaked onto Brucella Agar containing 7% sheep blood. The antibiotic strips were added and then the plates were incubated at 37 °C in a micro-aerobic environment provided by the CampyGen system (Oxoid Ltd) for another 3–4 days. The antibiotics’ susceptibility (S) and resistance (R) were recorded for each of the antibiotics previously listed.
In the histopathology laboratory, the paraffin-embedded tissue blocks were sectioned at 4 µm thickness and stained with the routine H and E stain for morphology, while modified Giemsa and Warthin-Starry stains were used for the identification of H. pylori. Sections found to show spiral bacteria in the mucosal layer or on the surface of the gastric epithelial cells were considered positive for H. pylori as assessed independently by two histopathologists.
In the histopathology laboratory, the paraffin-embedded tissue blocks were sectioned at 4 µm thickness and stained with the routine H and E stain for morphology, while modified Giemsa and Warthin-Starry stains were used for the identification of H. pylori. Sections found to show spiral bacteria in the mucosal layer or on the surface of the gastric epithelial cells were considered positive for H. pylori as assessed independently by two histopathologists.
10
Cultivation and Infection of C. jejuni Strains
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The C. jejuni wild-type (wt) strain 81-176 and its isogenic knockout mutant C. jejuni ΔhtrA, the complemented mutant ΔhtrA/htrA and protease-inactive S197A point mutation in the htrA gene were used throughout this study (Boehm et al., 2012 (link); Boehm et al., 2015 (link)). Bacterial cells were cultured using Campylobacter blood-free selective agar base including Campylobacter growth supplement provided by Oxoid (Wesel, Germany). Alternatively, the bacteria were grown on Mueller-Hinton (MH) agar supplemented with chloramphenicol (20 μg/ml) or kanamycin (30 μg/ml), respectively. Incubation was for 48 h at 37°C in jars using microaerobic conditions provided by the CampyGen™ system from Oxoid. All C. jejuni strains were harvested using sterile cotton swabs and resuspended in liquid BHI medium. The optical density (OD) was measured at 600 nm in an Eppendorf spectrophotometer to calculate the number of bacterial cells followed by host cell infection of C. jejuni using a multiplicity of infection (MOI) of 100.
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