Bacterial wild strains, S. Enteritidis (3546/6 2012) isolated from an environmental swab of a layer hens’ pen, S. Hadar (2507/5 2009) isolated from a broiler chicken cloacal swab and S. Senftenberg (3014/3 2012) isolated from an environmental swab of a layer hens’ pen, were obtained from the collection of pathogenic microorganisms of the OIE Reference Laboratory for Salmonellosis (Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy). Salmonella strains were stored in MicrobankTM (Pro-Lab Diagnostics, USA) at -80°C until needed. Klebsiella pneumoniae clone ST258 (García-Fernández et al., 2012 (link)), used as positive control, was kindly provided by Dr Alessandra Carattoli at Istituto Superiore di Sanità, Rome, Italy. The stock cultures were subcultured on tryptone agar (TA) slants at 4°C, and Salmonella serovars were confirmed by serotyping according to Grimont and Weill (2007) , transferred to 15 ml of Mueller-Hinton broth (MHB) and incubated at 37°C overnight.
Microbank
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The Microbank is a storage and transportation system for bacterial isolates. It is designed to maintain the viability of microorganisms during storage and shipment.
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Lab products found in correlation
Sabouraud dextrose agar, by Thermo Fisher Scientific (4 mentions)
Maldi biotyper, by Bruker (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Vitek 2, by bioMérieux (3 mentions)
Mrs broth, by Thermo Fisher Scientific (2 mentions)
Blood agar, by Thermo Fisher Scientific (2 mentions)
Generbox microaer, by bioMérieux (2 mentions)
Columbia agar containing 5 cattle blood, by Thermo Fisher Scientific (2 mentions)
Iq check l monocytogenes 2 kit, by Bio-Rad (1 mentions)
Maldi tof ms, by Shimadzu (1 mentions)
Mueller hinton agar, by Thermo Fisher Scientific (1 mentions)
Vitek ms, by bioMérieux (1 mentions)
Densichek, by bioMérieux (1 mentions)
Eb buffer, by Qiagen (1 mentions)
Proteinase k, by Avantor (1 mentions)
Quant it dsdna hs kit, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Dna rna shield, by Zymo Research (1 mentions)
Innova 44, by Eppendorf (1 mentions)
0.22 m filter, by Merck Group (1 mentions)
80 protocols using microbank
1
Salmonella Serovars Isolation and Identification
2
Isolation and Characterization of L. monocytogenes
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The isolation and characterization of L. monocytogenes were carried out according to EN ISO 16140 and UNI EN ISO 11290-1:2005. Briefly, the sponges were resuspended in 100 ml of Half Fraser Broth (HFB) and incubated at 30°C. After 24 h, 1.5 ml of each sample was collected and used for L. monocytogenes detection using the Bio-Rad IQ-Check- L. monocytogenes II kit (Biorad, USA). Positive samples were plated on ALOA agar and on Oxford agar media. A number of five colonies with the typical L. monocytogenes appearance were tested for haemolysis on Blood agar medium and finally tested by Christie Atkins Munch-Petersen test (CAMP test). Positive colonies were confirmed by Bio-Rad IQ-Check- L. monocytogenes II kit (Biorad, USA). Confirmed colonies were stored at −80°C in MicrobankTM (Pro-Lab Diagnostics, US).
3
Cultivating and Inactivating Aeromonas salmonicida
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The Aeromonas. salmonicida ssp. salmonicida wild-type strain JF 2267 used for all stimulation trials was kindly provided by J. Frey, University of Bern, Switzerland. The bacteria were cultivated from cryoconserved batches (MicrobankTM, PRO-LAB Diagnostics) on casein-peptone soymeal-peptone (CASO) slant agar (lysogeny broth (LB); SIFIN) at 15 °C for 98 h and subsequent mass culture replant in LB broth (SIFIN) for another 48 h. The initial cultures were checked for purity by Gram staining, cell morphology and motility. The bacterial suspension was concentrated by a 10-min centrifugation step at 4000 g at 4 °C. The supernatant was discarded and the bacterial pellet was washed once in sterile 0.9% sodium chloride solution and diluted to 1 x 108 bacteria/mL. Each dose was controlled afterwards by counting the colony-forming units (CFU) after culturing the bacteria on CASO agar plates at 15 °C.
For stimulation experiments, bacteria were inactivated in 1.5% paraformaldehyde for 1 h. The suspension of inactivated A. salmonicida (iA.s.) was adjusted to a concentration of 1.6 × 109 cells/mL, frozen in 1 mL aliquots and kept at −20 °C. Prior to use, the bacteria were thawed and diluted to the concentration of 5 × 107 cells/mL in sterile 1x PBS.
For stimulation experiments, bacteria were inactivated in 1.5% paraformaldehyde for 1 h. The suspension of inactivated A. salmonicida (iA.s.) was adjusted to a concentration of 1.6 × 109 cells/mL, frozen in 1 mL aliquots and kept at −20 °C. Prior to use, the bacteria were thawed and diluted to the concentration of 5 × 107 cells/mL in sterile 1x PBS.
4
Isolation of Beneficial Skin Microbiome
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Skin swabs were collected from the distal part of the limbs and other regions (pastern, the middle line of the chest, the pectoral area) of the body of 15 clinically healthy broodmares of Norik breed Muráň Plain type. The horses were kept on pasture in the Center of Horse breeding in Dobšiná, Slovak Republic. The swabs were placed into 1 mL of isotonic saline solution, and a series of 10-fold dilutions was prepared. From the appropriate dilutions, 0.1 mL aliquots were spread onto de Man-Rogosa-Sharpe agar (MRS, pH 6.4; Carl Roth GmbH + Co., KG, Karlsruhe, Germany) and cultivated under aerobic and anaerobic conditions (Gas Pak Plus, BBL, Microbiology Systems, Cockeysville, MD, USA) at 27 °C for 48–72 h. The morphologically different colonies were reclaimed from the individual dilutions under the same growth conditions to obtain pure cultures. The isolates were selected based on gram-positive staining and coccoid or rod shapes. The selected isolates were preserved in MicrobankTM (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at −70 °C.
5
Surveillance of Antibiotic-resistant Bacteria in China
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Fresh fecal samples were collected from patients for antibiotic-resistance surveillance at a tertiary class-A hospital in Beijing, China, from June to September 2019 (Table S3 ). The fresh samples were homogenized in PBS (pH = 7.2). The 100 μL sample of homogenate was then mixed with 10 mL of LB broth (supplemented with 2 mg/L tigecycline) and incubated for 12 h at 37 °C with 200 rpm shaking. Next, the enriched broth was streaked on CHROMagarTM Orientation agar plates with tigecycline (2 mg/L) and incubated at 37 °C for 24 h. Purified colonies were obtained after re-streaking three times on a MacConkey agar plate and were then stored in a MicrobankTM (Pro-Lab Diagnostics, Toronto, ON, Canada) at −70 °C. Bacterial species were identified using a MALDI-TOF/MS (Shimadzu, Kyoto, Japan) and reconfirmed by 16S rRNA gene sequencing. A colony PCR was used to screen tet(X)-positive clones using the universal primers, tet(X)-F (5′-TGA ACC TGG TAA GAA GAA GTG-3′) and tet(X)-R (5′-CAG ACA ATA TCA AAG CAT CCA-3′), and Sanger sequencing was used to confirm all amplicons after PCR amplification.
6
Bacterial Strains in Food and Dental Research
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The bacterial strains used in this study were as follows: foodborne pathogenic bacteria Bacillus cereus KCTC 3624, Listeria monocytogenes KCTC 13064, Staphylococcus aureus subsp. aureus KCTC 3881, Aeromonas hydrophila subsp. hydrophila KCTC 2358, Escherichia coli KCTC 2441, and Salmonella typhimurium KCCM 11862. They were maintained in cryogenic bead vials (MicrobankTM; Pro-Lab Diagnostics Inc., Toronto, ON, Canada) at −70 °C and activated in brain heart infusion (BHI) broth (Difco; Becton Dickinson, Spark, MD, USA) at 35 °C in an aerobic atmosphere condition for 16–20 h before use; dental plaque bacteria Enterococcus faecalis KCTC 5289, Streptococcus mutans KCTC 5458, Streptococcus mutans KCCM 40105, Streptococcus oralis KCCM 41567, Streptococcus sobrinus KCTC 5809, Streptococcus sobrinus KCCM 11,898, and Streptococcus sanguinis KCTC 5643 were maintained in cryogenic bead vials at −70 °C and activated in BHI broth at 35 °C in a 20% CO2 atmosphere condition for 16–20 h; lactic acid bacteria Lactobacillus acidophilus KCTC 3164 and Streptococcus thermophilus KCTC 3658 were maintained in cryogenic bead vials at −70 °C and activated in de Man Rogosa and Sharpe (MRS) agar (Difco) at 35 °C in a 20% CO2 atmosphere condition for 16–20 h.
7
Multidrug-Resistant P. aeruginosa Isolates Characterization
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Of the 721 MDR P. aeruginosa sent to CFASS for extended antimicrobial susceptibility testing over a 20-year period (2001–2020), one-tenth of the patient-unique isolates were randomly selected for this study. These isolates, stored in the bacterial preservation system MICROBANKTM (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at −80 °C, were plated onto Mueller-Hinton (MH) agar (Oxoid Ltd., Basingstoke, UK) and were identified using a Vitek® MS (BioMérieux, Inc., Durham, NC, USA). Isolates were tested by CFASS and were classified as multidrug-resistant if there was acquired non-susceptibility to at least one agent in the ≥3 antimicrobial group [22 (link)]. The media used for minimum inhibitory concentration (MIC) and synergy testing included Mueller-Hinton agar and cation-adjusted Mueller-Hinton II broth (CAMHB). P. aeruginosa ATCC 27853 was used as a quality control strain to validate the MIC values.
8
Evaluating ClO2 Gas Antibacterial Efficacy
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Two organisms were selected to test antibacterial activities of ClO2 gas:
Staphylococcus aureus ATCC 12600 and Escherichia coliATCC 11775. Each bacterium was bound to porous beads (MicrobankTM, Pro-Lab
Diagnostics, Round Rock, TX, USA) and stored at −80°C. The inoculated beads were streaked
onto blood agar plates and incubated at 37°C for 24 h. Each colony was diluted in heart
infusion broth (Eiken Chemical Co., Ltd., Tokyo, Japan) and adjusted to about 9 ×
108 colony forming units per ml (CFU/ml) by turbidity measurements
(DensiCHEK, bioMerieux Inc., Durham, NC, USA). Eighty microliters of each suspension was
inoculated onto a 1-cm paper disc (Advantec Co., Ltd., Tokyo, Japan) and then dried inside
a biosafety cabinet before use.
Commercially available biological indicator strips preloaded with>106spores of Bacillus atrophaeus ATCC 9372 (ACE test, Fukuzawa Shoji Co.,
Ltd., Yokohama, Japan) were also used to validate ClO2 gas
decontaminations.
Staphylococcus aureus ATCC 12600 and Escherichia coliATCC 11775. Each bacterium was bound to porous beads (MicrobankTM, Pro-Lab
Diagnostics, Round Rock, TX, USA) and stored at −80°C. The inoculated beads were streaked
onto blood agar plates and incubated at 37°C for 24 h. Each colony was diluted in heart
infusion broth (Eiken Chemical Co., Ltd., Tokyo, Japan) and adjusted to about 9 ×
108 colony forming units per ml (CFU/ml) by turbidity measurements
(DensiCHEK, bioMerieux Inc., Durham, NC, USA). Eighty microliters of each suspension was
inoculated onto a 1-cm paper disc (Advantec Co., Ltd., Tokyo, Japan) and then dried inside
a biosafety cabinet before use.
Commercially available biological indicator strips preloaded with>106spores of Bacillus atrophaeus ATCC 9372 (ACE test, Fukuzawa Shoji Co.,
Ltd., Yokohama, Japan) were also used to validate ClO2 gas
decontaminations.
9
Microbial DNA Extraction and Sequencing
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Pure cultures of each strain were grown in plates or broth. Cells were harvested and suspended in a tube with cryopreservative (MicrobankTM, Pro-Lab Diagnostics UK, London, UK) or with DNA/RNA Shield (Zymo Research, Irvine, CA, USA) following MicrobesNG strain submission procedures.
First, 5 to 40 μL of the suspension were lysed with 120 μL of TE buffer containing lysozyme (final concentration 0.1 mg/mL) and RNase A (ITW Reagents, Barcelona, Spain) at a final concentration 0.1 mg/mL, incubated for 25 min at 37 °C. Proteinase K (VWR Chemicals, Aurora, OH, USA) at a final concentration of 0.1 mg/mL and SDS (Sigma-Aldrich, St. Louis, MI, USA) at a final concentration of 0.5% v/v were added and incubated for 5 min at 65 °C. Genomic DNA was purified using an equal volume of SPRI beads and resuspended in EB buffer (Qiagen, Hilden, Germany).
DNA was quantified with the Quant-iT dsDNA HS kit (ThermoFisher Scientific, Waltham, MA, USA) assay in an Eppendorf AF2200 plate reader (Eppendorf UK Ltd., Stevenage, UK). Extracted DNA was eluted in 10 mM Tris-HCl pH 8.0 or nuclease free water and sent to MicrobesNG for sequencing.
First, 5 to 40 μL of the suspension were lysed with 120 μL of TE buffer containing lysozyme (final concentration 0.1 mg/mL) and RNase A (ITW Reagents, Barcelona, Spain) at a final concentration 0.1 mg/mL, incubated for 25 min at 37 °C. Proteinase K (VWR Chemicals, Aurora, OH, USA) at a final concentration of 0.1 mg/mL and SDS (Sigma-Aldrich, St. Louis, MI, USA) at a final concentration of 0.5% v/v were added and incubated for 5 min at 65 °C. Genomic DNA was purified using an equal volume of SPRI beads and resuspended in EB buffer (Qiagen, Hilden, Germany).
DNA was quantified with the Quant-iT dsDNA HS kit (ThermoFisher Scientific, Waltham, MA, USA) assay in an Eppendorf AF2200 plate reader (Eppendorf UK Ltd., Stevenage, UK). Extracted DNA was eluted in 10 mM Tris-HCl pH 8.0 or nuclease free water and sent to MicrobesNG for sequencing.
10
Phage Propagation and Purification
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Host bacterial isolates were plated on DifcoTM nutrient agar (NA) (BD, Sparks, Maryland, USA) from frozen stock (MicrobankTM, ProLab Diagnostics, Richmond Hill, ON, Canada), and then the phage were propagated as previously described [13 (link)]. Briefly, two bacterial cultures were prepared in DifcoTM nutrient broth (NB) (BD, Sparks, MD, USA): 0.9 mL at 108 CFU/mL in a 2 mL microcentrifuge tube and 100 mL at 106 CFU/mL in a 250 mL beveled flask. A 100 µL aliquot of phage stock solution (~108 PFU/mL) was then added to the 0.9 mL culture. Both cultures were incubated at 27 °C (165 rpm) in a Innova 44 shaking incubator (New Brunswick Scientific, Edison, NJ, USA). After 4 h, the contents of the 2 mL tube were transferred into the beveled flask and incubation continued overnight. The phage solution was then treated with chloroform, centrifuged, and passed through a 0.22 µm filter (Millipore, Billerica, MA, USA). The resulting phage stocks were stored in amber vials (Wheaton Industries, Millville, NJ, USA) with 1 mL of chloroform at 4 °C. All phages and bacteria hosts used in this study can be found in Table 2 .
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