The identification of clinical isolates was carried out on a Vitek2 Compact (BioMerieux) and later confirmed by sequencing of 80 bp of the variable regions V1 and V3 of the 16S rRNA gene by Pyrosequencing (PSQ96RA, Diatech). All strains were stored at −80°C in brain heart infusion (BHI, Merck) broth enriched with 10% glycerol (Sigma Aldrich) until testing. Before the in vitro experiments, the bacteria were thawed and reconstituted in tryptic soy agar (Biomérieux) for 24 h at 37°C. Prevotella bivia was grown on Schaedler agar (Oxoid) plus 3% defibrinated sheep blood (Thermofisher) and incubated in anaerobiosis for at least 48 h.
Schaedler agar
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United Kingdom, United States
Schaedler agar is a microbiological culture medium used for the isolation and cultivation of anaerobic bacteria. It provides the necessary nutrients and growth factors to support the growth of a wide range of anaerobic microorganisms.
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Lab products found in correlation
Chocolate agar, by Thermo Fisher Scientific (5 mentions)
Maldi tof ms, by Bruker (2 mentions)
Pseudomonas selective cetrimide agar, by Thermo Fisher Scientific (2 mentions)
Columbia blood agar, by Thermo Fisher Scientific (2 mentions)
Maldi biotyper, by Bruker (2 mentions)
Defibrinated sheep blood, by Thermo Fisher Scientific (1 mentions)
Tryptic soy agar, by bioMérieux (1 mentions)
Glycerol, by Merck Group (1 mentions)
Vitek 2 compact, by bioMérieux (1 mentions)
Columbia agar with 5 sheep blood, by BD (1 mentions)
Thioglycolate and schaedler broth, by bioMérieux (1 mentions)
Vitek ms, by bioMérieux (1 mentions)
Schaedler agar, by bioMérieux (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Generbag microaer, by bioMérieux (1 mentions)
Vitek 2 system, by bioMérieux (1 mentions)
Bact alert, by bioMérieux (1 mentions)
Anerogen, by Thermo Fisher Scientific (1 mentions)
Schaedler medium, by Thermo Fisher Scientific (1 mentions)
Microtiter plate, by Greiner (1 mentions)
15 protocols using schaedler agar
1
Bacterial Identification and Preservation
2
Periprosthetic Tissue Pathogen Identification
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Periprosthetic tissue samples were minced into pieces and homogenized in an Ultra-Turrax Drive control disperser (IKA®-Werke GmbH & Co. KG, Germany) at 6,000 rpm for 2 min in interval direction change. Briefly, the homogenized samples were inoculated on agar plates: Columbia agar with 5% sheep blood (Becton Dickinson, Heidelberg, Germany), chocolate agar and Schaedler agar (Oxoid, Munich, Germany) under aerobic conditions with 5% CO2 and anaerobically at 35± 1°C. Additionally, the samples were inoculated in thioglycolate and Schaedler broth (bioMérieux, Marcy L’Etoile, France) at 35 ± 1°C for 14 days. The identification of pathogens was performed by MALDI-TOF MS (VITEK® MS, bioMérieux, Marcy L’Etoile, France).
3
Identification of H. pylori from Biopsy
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Samples were tested for the presence of urease with the use of RUT—Rapid Urease Test (CLO test Kimberly-Clark) and placed in tubes with 0.9% NaCl solution and transported to the Department of Pharmaceutical Microbiology with Laboratory for Microbiological Diagnostics, Medical University of Lublin, Poland. Biopsies were smashed with the use of two microscope slides and spread onto Schaedler agar (BioMerieux, Marcy-l’Étoile, France) with 5% sheep blood plates and Schaedler agar with 5% sheep blood plates additionally supplemented with DENT (Oxoid), incubated for 3–7 days at 35 °C in microaerophilic (5–10% CO2, 80–90% N2, 5–10% O2, Generbag microaer, BioMerieux) conditions. H. pylori isolates were identified by colony morphology, Gram-staining and positive catalase (3% H2O2), urease and oxidase (BBL DrySlide Oxidase—Becton Dickinson, Franklin Lakes, NJ, USA) tests.
DNA of biopsy sample and bacterial isolates were extracted using QIAGEN QIAamp DNA Mini Kit (Qiagen, USA) according to the manufacturer’s instructions. The identification in biopsy samples and isolates were further confirmed as H. pylori using ureC gene amplification with specific primers [14 (link)]. Extracted DNA was frozen to −70 °C until its use.
DNA of biopsy sample and bacterial isolates were extracted using QIAGEN QIAamp DNA Mini Kit (Qiagen, USA) according to the manufacturer’s instructions. The identification in biopsy samples and isolates were further confirmed as H. pylori using ureC gene amplification with specific primers [14 (link)]. Extracted DNA was frozen to −70 °C until its use.
4
Blood and Bile Culture Protocols
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Samples for blood cultures (BC) were collected before or immediately after the initiation of antibiotic treatment by the bedside inoculation of 10 ml of blood into BC bottles (BacT/Alert, bioMérieux, Durham, NC, USA). Bottles were incubated at 37°C until microbial growth was detected or for at least seven days. Bile cultures were obtained via endoscopic retrograde cholangiopancreatography (ERCP) using catheter aspiration. Primary of bile cultures, as well as all subcultures, including those of positive BC cultures, were performed using standard solid media, e.g., Columbia blood agar (SIFIN, Berlin, Germany) for aerobic bacteria and Schaedler agar (Oxoid, Basingstoke, UK) for anaerobic bacteria. The cultivated microorganisms were identified and antibiotic susceptibility testing (MIC) was performed using a VITEK 2 system (bioMérieux, Durham, NC, USA), with the results interpreted according to the DIN- and EUCAST guidelines [20 (link)]
5
Anaerobic bacterial culture protocol
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The culture medium used for each strain is given in Supplementary Table 1 and included the following: Brain Heart Infusion with Inulin (BHII Agar): BHI Agar (BD Difco, cat. number 279830, Franklin Lakes, NJ, USA) supplemented with inulin from chicory (Sigma, cat. number I2255, Darmstadt, Germany), 1 g/l. Schaedler Agar (Oxoid, cat. number CM0497, Ireland), and modified Gifu Anaerobic Broth (mGAM) (HiMedia, cat. number M2079). All media were prepared according to manufacturer instructions and supplemented with 15 g agar/l whenever appropriate. Bacteria were cultured under strictly anaerobic conditions using an anaerobic workstation (Anaerobic chamber; Coy Laboratories, Ann Arbor, MI, USA) containing a gas mixture of 10% CO2, 5% H2, and 85% N2. Bacteria were incubated at 37°C for 3–4 days before MALDI-TOF MS analysis. Culture conditions are summarized in Supplementary Table 1 (strains used for library construction) and Supplementary Table 2 (validation strains).
6
Quantifying Bacterial Translocation
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Bacterial translocation was assessed in samples of blood, liver and MLN. Samples were homogenized in buffered peptone water (1g/ml) and 100 μl of the resulting homogenates were inoculated in plates for bacterial counting, using Schaedler agar (Oxoid, UK) for Bacteroides and Wilkins- Chalgren anaerobe agar (Oxoid, agar) for total anaerobe quantification after incubation at 37°C in anaerobic conditions (AneroGen; Oxoid, Basingstoke, UK) for 3 days. The results are expressed as incidence of translocation in the event of positive growth on agar plates, even a single colony of any microorganism. Data of CFU/ g tissue are also given.
7
Determining Bactericidal Efficacy of Silver(I) Complexes
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To assess the susceptibility of oral pathogenic bacteria to silver(I) complexes, minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) were determined. For this purpose, test agents were dissolved in SCHAEDLER medium (Oxoid, Germany) to obtain a concentration of 40 mM. Serial dilution was performed for all compound solutions. Afterwards, 100 μL of each bacterial suspension (see 2.2) were pipetted into wells of microtiter plates (Greiner, Germany) containing 100 μL of the corresponding diluted compound solution. After incubation at 37 °C for 24 h, the bacterial growth was evaluated. The MIC represents the dilution stage in which no clouding of the test specimen was observed. Pure SCHAEDLER medium served as positive control, SCHAEDLER medium including the same amount of the bacterial suspension as negative control. To distinguish MCI from MBC, 100 μL of each compound solution with concentrations below MIC were spread onto a Petri dish with SCHAEDLER agar (Oxoid, Germany) and cultivated under identical conditions. The concentration of the dilution series where no growth of bacteria was observed was considered as MBC.
8
Identification and Antimicrobial Susceptibility of Blood Isolates
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Positive blood culture bottles were subjected to Gram staining, and subcultures were streaked onto Columbia agar with 5 % sheep blood and Mac Conkey agar, Schaedler agar was used for anaerobic growth and Sabouraud agar for cultivation of fungi (all from Oxoid). After overnight growth, microorganisms were identified to species level using MALDI-TOF (Bruker Daltonics, Bremen, Germany) or biochemical profiling (Vitek2 XL, Biomerieux, Marcy L′Etolie, France). Susceptibility testing was carried out using a semi-automated system (Vitek 2, Biomerieux) or Kirby-Baur disc diffusion assays. Breakpoints to differentiate between susceptible and non-susceptible phenotypes were applied according to the European Committee on Antimicrobial Susceptibility Testing.
9
Periprosthetic Tissue Culture Protocol
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The homogenized periprosthetic tissue samples were applied for cultivation onto sheep-blood agar and chocolate agar (Oxoid, Basingstoke, Hampshire, United Kingdom). For anaerobic cultures, Schaedler agar, Schaedler KV agar (Oxoid, Basingstoke, Hampshire, United Kingdom) and Columbia blood agar (biomerieux, Marcy l’Etoile, France) were inoculated and incubated for five days. All specimens were also incubated for fourteen days using brain-heart infusion broth (BHI, Oxoid, Basingstoke, Hampshire, United Kingdom) and thioglycollate broth medium additionally incorporated with liver digest and finally supplemented with hemin and horse serum (LT, SIFIN, Berlin, Germany). For more details about this approach, see the literature [2 (link), 3 (link)]. As an adjunct, the results of all investigated prostheses and components had no influence on the study and are therefore not discussed in more detail here.
The organisms were identified by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; BrukerDaltonics, Bremen, Germany) using the direct transfer method according to the recommendation of the manufacturer.
The organisms were identified by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; BrukerDaltonics, Bremen, Germany) using the direct transfer method according to the recommendation of the manufacturer.
10
Antimicrobial Efficiency of Silver(I) Complexes
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To evaluate the antimicrobial concentration-related efficiency of the silver(I) complexes, agar diffusion tests were performed. The general procedure of this test was described previously in the literature [21 (link)]. Briefly: 100 μL of each bacterial suspension (108 bacteria/mL) were pipetted and spread onto a Petri dish with Schaedler agar (Oxoid, Germany). 100 μL of each test fluid were then filled into a central hole (diameter: 8 mm) of each Petri dish. After bacteria specific incubation time (>48 h), the diameters of the inhibition zones were measured. For positive (negative) control chlorhexidine (distilled water) was used. Six specimens were prepared for each test fluid (AgNO3; complex 3 and 4 ) with two different concentrations (10 mM and 20 mM).
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