Seventy-nine A. butzleri and 6 A. cryaerophilus cultures were isolated from meat samples, purchased from retail market in Lower Silesia region (Poland) as described previously [22 (link)]. Among 79 A. butzleri isolates, 58, 11, and 10 were obtained from chicken, beef, and pork samples, respectively. Within A. cryaerophilus cultures, 3 isolates were from beef, 2 were from chicken, and one was from pork meat. Arcobacter spp. isolates were cultured according to Houf et al. [23 (link)] with modifications, in Arcobacter broth (Oxoid) with selective supplement containing cefoperazone, amphotericin B, and teicoplanin (CAT, Oxoid). Additionally, novobiocin (32 mg/L), 5-fluorouracil (100 mg/L), and trimethoprim (64 mg/L) (Sigma) were added to the broth. After 48-hour incubation in aerobic atmosphere, at 30°C, the bacteria were subcultured on Arcobacter agar plates (supplemented with chemotherapeutics mentioned above) and in parallel on agar plates with defibrinated sheep blood (Oxoid). Phenotypically suspected colonies were transferred to blood agar plates and incubated in aerobic conditions for 48 h at 30°C. One Arcobacter spp. isolate per sample was taken for further characterization. The isolates were preserved by freezing in Cryobank (Mast Diagnostics) at −80°C.
Arcobacter broth
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United Kingdom
Arcobacter broth is a microbiological growth medium used for the cultivation and isolation of Arcobacter species, which are bacteria commonly found in the environment and associated with foodborne illnesses. The broth provides the necessary nutrients and growth conditions for the proliferation of Arcobacter organisms.
Automatically generated - may contain errors
Lab products found in correlation
Defibrinated sheep blood, by Thermo Fisher Scientific (1 mentions)
Novobiocin, by Merck Group (1 mentions)
Trimethoprim, by Merck Group (1 mentions)
Cryobank, by Mast Diagnostics (1 mentions)
5 fluorouracil, by Merck Group (1 mentions)
Ivd bacterial test standard, by Bruker (1 mentions)
High pure pcr template preparation kit, by Roche (1 mentions)
Biotyper 3, by Bruker (1 mentions)
Norfloxacin, by Thermo Fisher Scientific (1 mentions)
Amoxicillin, by Thermo Fisher Scientific (1 mentions)
Tetracycline, by Thermo Fisher Scientific (1 mentions)
Ciprofloxacin, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Cefotaxime, by Thermo Fisher Scientific (1 mentions)
Mueller hinton agar plate, by Merck Group (1 mentions)
Erythromycin, by Thermo Fisher Scientific (1 mentions)
Ae elution buffer, by Qiagen (1 mentions)
Nanodrop 2000 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
5 protocols using arcobacter broth
1
Isolation and Identification of Arcobacter spp. from Retail Meat
2
Isolation and Identification of Aliarcobacter from Fecal Samples
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The Aliarcobacter isolates from fecal samples were cultivated in Arcobacter broth (Oxoid GmbH, Wesel, Germany) which was supplemented with three different antibiotics (cefoperazone, amphotericin, and teicoplanin (CAT), Oxoid GmbH). The broth was then spread on plates (Mueller–Hinton agar/CAT/5% defibrinated bovine blood, Sifin GmbH, Berlin, Germany). The incubation criteria for each step were: 48–72 h, 30 °C and microaerophilic atmosphere (5% O2, 10% CO2, and 85% N2). Suspicious colonies were further cultivated and then identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as described before [32 (link),33 (link)]. IVD Bacterial Test Standard and Biotyper 3.1 software were used (Bruker Daltonik GmbH, Bremen, Germany). DNA was purified using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s instructions, and the species identification was confirmed with a multiplex PCR assay [34 (link)].
3
Arcobacter broth DNA Isolation and Antibiotic Susceptibility
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Total DNA was isolated as described by Agersborg et al. (1997) . Briefly, the bacteria from 1 ml overnight culture in Arcobacter broth (Oxoid) were pelleted by centrifugation and suspended in 200 μl of distilled water containing 1% Triton X-100. The mixture was boiled for 10 min and then the tubes were centrifuged for 5 min at 13 000 × g. The supernatant containing DNA was used in PCR. (3) 2 (100) 2 (100) 2 (100) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Chicken meat/70 A. butzleri 10 (14) 7 (70) 7 (70) 8 (80) 4 (40) 4 (40) 9 (90) 5 (50) 5 (50 67)
Total/210 * A -ampicillin (10 μg/disc), AC -amoxicillin with clavulonic acid (20/10 μg/disc), C -cefotaxime (30 μg/disc), T -tetracycline (30 μg/disc), G -gentamicin (10 μg/disc), , E -erythromycin (15 μg/disc), CI -ciprofloxacin (5 μg/disc), N -norfloxacin (10 μg/disc).
Total/210 * A -ampicillin (10 μg/disc), AC -amoxicillin with clavulonic acid (20/10 μg/disc), C -cefotaxime (30 μg/disc), T -tetracycline (30 μg/disc), G -gentamicin (10 μg/disc), , E -erythromycin (15 μg/disc), CI -ciprofloxacin (5 μg/disc), N -norfloxacin (10 μg/disc).
4
Antibiotic Susceptibility of Arcobacter spp.
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Susceptibility of Arcobacter spp. isolates to ampicillin (10 μg/disc), amoxicillin with clavulonic acid (20/10 μg/disc), gentamicin (10 μg/disc), cefotaxime (30 μg/disc), tetracycline (30 μg/disc), erythromycin (15 μg/disc), ciprofloxacin (5 μg/disc) and norfloxacin (10 μg/disc) (all substances from Oxoid Ltd., United Kingdom) was tested by the disk-diffusion method. Briefly, the isolates were grown aerobically in Arcobacter broth (Oxoid) at 30 o C for 48 h. After cultivation, a suspension of bacteria was prepared in physiological saline and the turbidity of inoculum was adjusted to McFarland 0.5. Bacteria were plated onto Mueller-Hinton agar plates (Merck). Thereafter, disks were placed onto the agar and the plates were incubated in aerobic atmosphere at 30 o C for 48 h.
To date, no recommendation of breakpoints values for Arcobacter spp. are available, thus according to Shah et al. (2012) classification of isolates as resistant or susceptible was based on breakpoints values recommended for Enterobacteriaceae (CLSI 2010). Reference E. coli ATCC 25922 strain served as control.
To date, no recommendation of breakpoints values for Arcobacter spp. are available, thus according to Shah et al. (2012) classification of isolates as resistant or susceptible was based on breakpoints values recommended for Enterobacteriaceae (CLSI 2010). Reference E. coli ATCC 25922 strain served as control.
5
Arcobacter spp. DNA Extraction and Purification
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DNA extraction and purification was performed as described by Mottola et al. (2016) .
Briefly, colonies identified as Arcobacter spp. were transferred onto Arcobacter broth (Oxoid, Basingstoke, UK) and incubated at 30 °C for 48 h. One millilitre of pure AB culture of presumptive Arcobacter spp. was centrifuged at 7500 rpm for 10 min at room temperature. DNA extraction and purification was performed with the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) and eluted with 80 μL AE Elution Buffer (QIAGEN, Hilden, Germany). The DNA concentration and purity were established by evaluating the ratio A260nm/A280nm using a NanoDrop 2000/2000c Spectrophotometer (Thermo Scientific, MA, USA).
Briefly, colonies identified as Arcobacter spp. were transferred onto Arcobacter broth (Oxoid, Basingstoke, UK) and incubated at 30 °C for 48 h. One millilitre of pure AB culture of presumptive Arcobacter spp. was centrifuged at 7500 rpm for 10 min at room temperature. DNA extraction and purification was performed with the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) and eluted with 80 μL AE Elution Buffer (QIAGEN, Hilden, Germany). The DNA concentration and purity were established by evaluating the ratio A260nm/A280nm using a NanoDrop 2000/2000c Spectrophotometer (Thermo Scientific, MA, USA).
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