The bacterial strains and plasmids used in this study are indicated in Table A1 (Appendix 1). For cultivation of bacteria, strains were routinely grown in LB medium (E. coli, P. putida) or M9 minimal salts agar containing 0.5% glucose (B. cenocepacia) at 37°C. M9 minimal salts contained 42 mM Na2HPO4, 22 mM KH2PO4, 19 mM NH4Cl, 9 mM NaCl, 1 mM MgSO4, and 0.1 mM CaCl2. Antibiotics were used, when appropriate, at the following concentrations: ampicillin (Ap), 100 μg/ml (E. coli); chloramphenicol (Cm), 25 μg/ml (E. coli, P. putida) and 50–100 μg/ml (B. cenocepacia); kanamycin (Km), 50 μg/ml (E. coli and B. cenocepacia); rifampicin (Rf), 100 μg/ml (E. coli and B. cenocepacia); and trimethoprim (Tp), 25 μg/mL (E. coli), 25 μg/ml (B. cenocepacia H111 and Pc715j), and 100 μg/ml (B. cenocepacia K56‐2). For selection of trimethoprim resistance in E. coli, Iso‐Sensitest Agar (Oxoid) was employed, and for selection of kanamycin resistance in B. cenocepacia, Lennox agar was utilized. Dialyzed brain‐heart infusion (D‐BHI) broth was prepared according to Sokol, Ohman, and Iglewski (1979 ) and used as the liquid growth medium for cultures of B. cenocepacia undergoing secreted protein extraction.
Iso sensitest agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Iso-Sensitest agar is a culture medium used for the isolation and cultivation of a wide range of microorganisms. It provides a standardized and reproducible growth environment for performing antimicrobial susceptibility testing.
Automatically generated - may contain errors
Lab products found in correlation
Antimicrobial gradient strips, by Thermo Fisher Scientific (2 mentions)
Glucose 6 phosphate (g6p), by Merck Group (2 mentions)
Etest, by bioMérieux (2 mentions)
Kanamycin, by Thermo Fisher Scientific (1 mentions)
Api 20ne, by bioMérieux (1 mentions)
E test method, by bioMérieux (1 mentions)
Mrs broth, by Avantor (1 mentions)
Api 20e system, by bioMérieux (1 mentions)
Vitek 2, by bioMérieux (1 mentions)
Epsilometer test, by bioMérieux (1 mentions)
Wilkins chalgren agar, by Thermo Fisher Scientific (1 mentions)
Api 20e strip, by bioMérieux (1 mentions)
Maldi tof, by Bruker (1 mentions)
Fosfomycin, by Merck Group (1 mentions)
Alafosfalin, by Merck Group (1 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (1 mentions)
Avibactam, by Merck Group (1 mentions)
Ertapenem, by Merck Group (1 mentions)
Ceftolozane, by Merck Group (1 mentions)
Mic test strip, by Liofilchem (1 mentions)
36 protocols using iso sensitest agar
1
Cultivation and Selection of Bacterial Strains
2
Detecting Metallo-Beta-Lactamase in P. aeruginosa
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P. aeruginosa was identified with conventional laboratory methods and/or an API 20 NE® instrument (BioMerieux, Lyon, France). Antimicrobial susceptibility was tested with disk diffusion on Iso-Sensitest Agar® (Oxoid Ltd., London, UK). Breakpoints established by the Swedish Reference Group for Antibiotics was used (nowadays, exchanged for the European Committee on Antimicrobial Susceptibility Testing).
The MBL phenotype was detected with the Etest for MBLs (AB Biodisk, Solna, Sweden). A reduction in imipenem minimum inhibitory concentrations (MICs) by ≥3 twofold dilutions in the presence of ethylenediaminetetraacetic acid (EDTA) was interpreted as being positive for MBL production. An additional double-disk test with imipenem ± EDTA and ceftazidime ± 2-mercaptopropionic acid (MPA) was performed as earlier described [22 (link),23 (link)]. The MICs of MBL-positive strains were further determined for imipenem, meropenem, piperacillin/tazobactam, ceftazidime, aztreonam, gentamicin, amikacin, tobramycin, ciprofloxacin, fosfomycin, and colistin.
The MBL phenotype was detected with the Etest for MBLs (AB Biodisk, Solna, Sweden). A reduction in imipenem minimum inhibitory concentrations (MICs) by ≥3 twofold dilutions in the presence of ethylenediaminetetraacetic acid (EDTA) was interpreted as being positive for MBL production. An additional double-disk test with imipenem ± EDTA and ceftazidime ± 2-mercaptopropionic acid (MPA) was performed as earlier described [22 (link),23 (link)]. The MICs of MBL-positive strains were further determined for imipenem, meropenem, piperacillin/tazobactam, ceftazidime, aztreonam, gentamicin, amikacin, tobramycin, ciprofloxacin, fosfomycin, and colistin.
3
Antibiotic Resistance Screening Protocol
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Strains were screened for resistance to antibiotics by agar disk diffusion on Iso-Sensitest media (Iso-Sensitest Agar, Oxoid, Basingstoke, UK). Zones of inhibition were evaluated using Mastring M13 and Mastring M48 according to manufacturers instructions (Mast Diagnostics, Merseyside, UK). The minimum inhibitory concentrations (MICs) to oxacillin were additionally evaluated using “M.I.C. evaluators,” antimicrobial gradient strips designed for accurate minimum inhibitory concentration (MIC) values (Oxoid Ltd., Basingstoke, UK). The categories susceptible, intermediate resistant, or resistant were assigned on the basis of the CLSI antimicrobial susceptibility testing standards.
4
Antibiotic Susceptibility Testing of LAB
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Minimum inhibitory concentrations were determined using an E-test (BioMerieux, Lyon, France) for nine antibiotics: tetracycline, erythromycin, streptomycin, gentamicin, clindamycin, ampicillin, kanamycin, chloramphenicol and vancomycin. Concentrations for all antibiotics were 0.016–256 μg/mL, except for streptomycin at 0.064–1024 μg/mL. The LAB isolates were diluted to final concentrations of 106–107 CFU/mL and then inoculated onto Iso-Sensitest agar (Oxoid, Basingstoke, UK) supplemented with MRS agar (Georgieva et al., 2015 (link)), and incubated at 37 °C for 24 h. Breakpoint values were interpreted according to EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) (2012) (Table S1 ).
5
Antibiotic Susceptibility Profiling
6
Antimicrobial Susceptibility Testing Protocol
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Susceptibility testing was performed by disc diffusion to representatives of beta-lactam and non-beta-lactam antimicrobial classes on ISO-Sensitest agar (Oxoid, Basingstoke, UK) and results were interpreted according to the BSAC (British Society for Antimicrobial Chemotherapy) interpretative criteria.25 (link)E. coli ATCC 25922 was used as control strain. All isolates were tested for ESBL production by the double disc synergy test (DDST).26 (link)
7
Antibiotic Susceptibility of LAB Strains
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The LAB strains were considered resistant or sensitive to each tested antibiotic (ampicillin, vancomycin, gentamycin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, chloramphenicol) according to breakpoints proposed by the European Food Safety Authority (EFSA) [36 ]. The minimum inhibitory concentration (MIC) was determined by the Etest® method (BioMérieux, Marcy l’Etoile, France), using the LAB susceptibility test medium (LSM) agar, consisting of a mixture of Iso-Sensitest agar (Oxoid) (90%) and MRS agar (10%) (pH 6.7), as recommended by ISO 10932/IDF 223 [37 ]. In detail, individual colonies, grown on LSM agar for 24 h at 37 °C, were suspended in a 0.85% NaCl solution to reach a McFarland standard value of 0.5, then were swabbed on LSM agar and dried before applying the Etest® strips. After incubation at 37 °C for 48 h, MIC values were determined following the manufacturer’s instructions. Strains showing MICs lower or higher than the EFSA’s breakpoints were considered as sensitive or resistant, respectively [36 ]. All analyses were performed in triplicate.
8
Antibacterial Efficacy of Silver-Doped Coacervate Glass
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The silver-doped coacervate glass (COA3) was investigated for its ability to inhibit
bacterial growth using a disk diffusion methodology (BSAC Disk Diffusion Method for
Antimicrobial Susceptibility Testing, Version 4, 2005). Isosensitest agar (Oxoid,
Basingstoke, UK) plates were inoculated with a standardized culture (optical density of
0.03 at a wavelength of 600 nm) of Psuedomonas aeruginosa(Epidemiological strain, School of Dentistry, University of Liverpool). One hundred
milligrams of both control (COA1) and silver-doped glass powders were pressed into 5 mm
diameter discs using Atlas™ Evacuable Pellet Dies (Specac Ltd, UK) and placed in each of
the inoculated plates. The experiment was conducted in triplicate and the glass not
containing any silver was used as a negative control. These plates were then incubated
aerobically at 37℃ for 24 h. The diameters of any zones that had formed around the samples
were measured using callipers.
bacterial growth using a disk diffusion methodology (BSAC Disk Diffusion Method for
Antimicrobial Susceptibility Testing, Version 4, 2005). Isosensitest agar (Oxoid,
Basingstoke, UK) plates were inoculated with a standardized culture (optical density of
0.03 at a wavelength of 600 nm) of Psuedomonas aeruginosa(Epidemiological strain, School of Dentistry, University of Liverpool). One hundred
milligrams of both control (COA1) and silver-doped glass powders were pressed into 5 mm
diameter discs using Atlas™ Evacuable Pellet Dies (Specac Ltd, UK) and placed in each of
the inoculated plates. The experiment was conducted in triplicate and the glass not
containing any silver was used as a negative control. These plates were then incubated
aerobically at 37℃ for 24 h. The diameters of any zones that had formed around the samples
were measured using callipers.
9
Antibiotic Susceptibility Testing for Wound Pathogens
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For primary wound pathogens and any silver and zinc-resistant isolates, the susceptibility to appropriate antibiotics was tested on IsoSensitest agar (Oxoid, Basing stoke, UK) [5 (link)]. All plates were incubated at 37°C for 18-24 h. For rapid and convenient identification of Enterococcus species and its vancomycin susceptibility, we used a SpectraTM VRE test. It is a selective and differential chromogenic medium, con-taining 6µg/mL of vancomycin, intended for use in the qualitative detection of vancomycin-resistant E. faecium and E. faecalis. According to the manufacturer’s protocols, we verified the Enterococcus species and vancomycin suscep-tibility.
10
Synthesis and Antimicrobial Activity of Silver Nanoparticles
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Carboxymethyl cellulose (CMC) and aminocellulose (AMC, 1%, DS = 0.5) were provided by the University of Jena and TITK, Germany. Silver nitrate (AgNO3, purity > 99%), sodium borohydride (NaBH4, purity > 96%), ethanol and powdered α-cellulose were purchased from Lancaster, Fluka, Fisher Scientific and Sigma Aldrich, respectively. Metal salts and other regents were used without further purification. Deionised water was used for all experiments. The antimicrobial activity of silver nanoparticles was assessed against meticillin-resistant Staphylococcus aureus (MRSA) (NCTC 12493) purchased from the Health protection agency laboratories, Porton Down, UK. MRSA was selected due to its often-high presence in hospital and community settings and its association with antibiotic resistance and potentially life-threatening infections. All agar and media including IsoSensitest agar and broth were purchase from Oxoid, UK.
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