A total of 40 A. butzleri strains (see Table S1 in the Supplemental Material ) were included in this study. All strains were collected and identified at the species level using molecular methods (multiplex polymerase chain reaction [PCR] and rpoB sequencing) during a previous Arcobacter prevalence study in Kaunas, Lithuania [20 (link)]. All 40 strains were also previously tested for susceptibility to six antimicrobial agents (ampicillin, ciprofloxacin, gentamicin, tetracycline, azithromycin, and erythromycin) using the gradient strip diffusion method [20 (link)]. Half of A. butzleri strains were isolated from different food products obtained from local retail markets: raw cow milk (n = 11), chicken meat (n = 7) and ready-to-eat (RTE) salad mixes (n = 2). The remaining strains originated from human stool (n = 10) and environmental water samples (n = 10, including lake and river-water samples). All 40 strains were stored at −80 °C in a brain–heart infusion broth (BHI) (Oxoid, Thermo Fisher Scientific, Basingstoke, UK) containing 30% (v/v) glycerol (Stanlab, Lublin, Poland). Before genomic DNA (gDNA) extraction, A. butzleri strains were cultured on Mueller–Hinton agar (Oxoid, Thermo Fisher Scientific) plates supplemented with 5% (v/v) defibrinated sheep blood (MHB) (Oxoid, Thermo Fisher Scientific) at 30 °C for 48 h under microaerobic conditions.
Glycerol
Manufactured by EKF Diagnostics
Sourced in Poland, United Kingdom
Glycerol is a clear, odorless, and viscous liquid. It is a polyol compound and is widely used in various industries, including the pharmaceutical and diagnostic sectors. Glycerol is a core component in numerous laboratory and diagnostic applications, where it serves as a stabilizing agent, solvent, and preservative.
Automatically generated - may contain errors
Lab products found in correlation
Blood agar base no 2, by Thermo Fisher Scientific (2 mentions)
Lactucin, by Extrasynthese (1 mentions)
Lactucopicrin, by Extrasynthese (1 mentions)
Lc ms grade solvents, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Chlorogenic acid, by Merck Group (1 mentions)
Malt extract agar, by Merck Group (1 mentions)
Cetrimide, by bioMérieux (1 mentions)
Vitek 2 compact system, by bioMérieux (1 mentions)
Macconkey, by bioMérieux (1 mentions)
6 protocols using glycerol
1
Isolation and Characterization of A. butzleri Strains
2
Potato Starch-Based Phytochemical Analysis
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Potato starch was purchased from PEPEES (Łomża, Poland); glycerol ≥ 99.5% from Stanlab (Lublin, Poland); phytic acid 50% water solution, LC-MS grade solvents (water ≥ 95%, methanol ≥ 99.9%, formic acid ≥ 98%), and chlorogenic acid ≥ 95% from Sigma-Aldrich (St. Louis, MO, USA); methanol analytical grade ≥ 99.8%, concentrated hydrochloric acid 37%, and Fehling, Carrez, and Folin-Ciocalteu reagents from Chempur (Piekary Śląskie, Poland); calcium chloride anhydrous ≥ 97% and natrium carbonate anhydrous ≥ 98% from Eurochem (Tarnów, Poland); lactucin ≥ 95% and lactucopicrin ≥9 5% from Extrasynthese (Genay, France); and Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), Sabouraud, and Malt Extract Agar (MEA) media from Merck (Darmstadt, Germany).
3
Multilocus Sequence Typing of C. jejuni
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A total of 341 C. jejuni strains of known Multilocus sequence types (MLST) were included in this study. The strain collection was composed of 41 strains isolated from dairy cattle at farm level, 98 strains isolated from retail broiler products, 101 strains isolated from wild birds and 101 strains from human clinical cases that were collected at the Microbiological Laboratory of Kaunas Clinical Hospital over one year period. The MLST method based on sequencing of seven housekeeping genes was applied for each of the isolates as described in a previous study (Ramonaite et al., 2014 (link), 2017 (link)). The strains were stored at -80°C in brain heart infusion broth (BHI) (Oxoid, Basingstoke, United Kingdom) with 30 % glycerol (Stanlab, Lublin, Poland).
4
Characterization of C. jejuni Isolates
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In total, seven C. jejuni isolates from bacterial culture collection of the Department of Food Safety and Quality of Lithuanian University of Health Sciences were tested in this study. These isolates were previously characterized by Multi Locus Sequence Typing (MLST) and assigned to CC179 and CC21 clonal complexes, with wide spread in Lithuania [14 (link),15 (link)]. The isolates were stored at −80 °C in brain heart infusion broth (BHI) (Oxoid, Basingstoke, UK) with 30% glycerol (Stanlab, Lublin, Poland). The isolates’ recovery were performed by plating the stocks on Blood agar base No. 2 (Oxoid, Basingstoke, UK) supplemented with 5% defibrinated horse blood (E&O Laboratories Limited, Scotland, UK) and further incubation under microaerophilic conditions (5% oxygen, 10% carbon dioxide, and 85% nitrogen) at 42 °C for 48 h.
5
Clinical Isolates of P. aeruginosa in CF
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The subject of the study was a collection of 120 P. aeruginosa strains isolated from 96 clinical materials obtained from 10 adults (≥ 18 years of age) suffering from CF, from January 2012 to December 2019. Table 1 presents the characteristics of the subjects.
The retrospective and prospective analysis of P. aeruginosa strains was approved by the Bioethics Committee of the Institute of Tuberculosis and Lung Diseases in Warsaw (No. KB-74/2019).
The materials were processed in accordance with the principles of applicable laboratory procedures, inoculated directly on agar media: Columbia supplemented with 5% sheep blood, MacConkey and Cetrimide (bioMérieux, Marcy-l'Étoile France). The cultures were incubated for 24 to 48 h at 37 °C. Preliminary identification of P. aeruginosa was performed by routine microbiological methods (oxidase, pigment production, growth at 42 °C) and confirmed with the commercial VITEK®2Compact system (bioMérieux). P. aeruginosa strains (dominant morphotypes) were stored at − 80 °C in the MicrobankTM system (Pro-Lab) or in 50% (v/v) glycerol (Stanlab, Poland) in the strain bank of the Department of Microbiology at the Institute of Tuberculosis and Lung Diseases in Warsaw.
The retrospective and prospective analysis of P. aeruginosa strains was approved by the Bioethics Committee of the Institute of Tuberculosis and Lung Diseases in Warsaw (No. KB-74/2019).
The materials were processed in accordance with the principles of applicable laboratory procedures, inoculated directly on agar media: Columbia supplemented with 5% sheep blood, MacConkey and Cetrimide (bioMérieux, Marcy-l'Étoile France). The cultures were incubated for 24 to 48 h at 37 °C. Preliminary identification of P. aeruginosa was performed by routine microbiological methods (oxidase, pigment production, growth at 42 °C) and confirmed with the commercial VITEK®2Compact system (bioMérieux). P. aeruginosa strains (dominant morphotypes) were stored at − 80 °C in the MicrobankTM system (Pro-Lab) or in 50% (v/v) glycerol (Stanlab, Poland) in the strain bank of the Department of Microbiology at the Institute of Tuberculosis and Lung Diseases in Warsaw.
6
Campylobacter jejuni Isolation and Identification
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A total of 292 C. jejuni isolates from infected children (n = 100), raw (n = 77), and marinated (n = 19) broiler products and wild birds: pigeons (n = 39) and crows (n = 57) were selected. Food and wild bird isolates were from the Campylobacter collection at the Department of Food Safety and Quality, Veterinary Academy, Lithuanian University of Health Sciences. The human C. jejuni isolates were received from the Microbiological laboratory of Kaunas Clinical Hospital and were isolated in the period from 2011 to 2012. Identification of Campylobacter isolates was performed with multiplex PCR as described by Wang et al. (2002 (link)) with the minor modifications described previously by Ramonaite et al. (2015 (link)).
Campylobacter isolates were stored as frozen stocks at −80°C in in brain heart infusion broth (BHI) (Oxoid Ltd., Basingstoke, UK) with 30% glycerol (Stanlab, Poland). They were recovered from frozen stocks on Blood agar base No. 2 (Oxoid, Basingstoke, Hampshire, England) supplemented with 5% defibrinated horse blood (E&O Laboratories, Burnhouse, Bonnybridge, Scotland) and incubated under microaerophilic conditions (5% oxygen, 10% carbon dioxide and 85% nitrogen) at 37°C for 48 h.
Campylobacter isolates were stored as frozen stocks at −80°C in in brain heart infusion broth (BHI) (Oxoid Ltd., Basingstoke, UK) with 30% glycerol (Stanlab, Poland). They were recovered from frozen stocks on Blood agar base No. 2 (Oxoid, Basingstoke, Hampshire, England) supplemented with 5% defibrinated horse blood (E&O Laboratories, Burnhouse, Bonnybridge, Scotland) and incubated under microaerophilic conditions (5% oxygen, 10% carbon dioxide and 85% nitrogen) at 37°C for 48 h.
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