Two bacterial strains were used in this study: Gram negative Klebsiella pneumoniae American Type Culture Collection (ATCC) 700603 producing extended-spectrum beta-lactamase (ESBL) and Gram positive Enterococcus faecalis ATCC 51299 presenting the vanB gene inducing glycopeptide resistance. The cultures were grown on Columbia agar with 5% sheep blood (Bio-Rad, Marnes-La-Coquette, France) and incubated for 24 h at 35 ± 2 °C.
Columbia agar
Manufactured by Bio-Rad
Sourced in France
Columbia agar is a general-purpose microbiological culture medium used for the isolation and cultivation of a wide range of bacterial species. It provides the necessary nutrients and growth factors to support the growth of diverse microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Sheep blood, by Bio-Rad (3 mentions)
Genbox system, by bioMérieux (1 mentions)
Anaerocult system, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Vitek ms instrument, by bioMérieux (1 mentions)
Columbia cna agar, by Bio-Rad (1 mentions)
Riboflavin, by Merck Group (1 mentions)
Mupirocin, by GlaxoSmithKline (1 mentions)
Anaerobic bag, by bioMérieux (1 mentions)
9 protocols using columbia agar
1
Profiling Antibiotic-Resistant Bacteria
2
Isolation and Identification of Cutibacterium acnes Strains
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Cutibacterium acnes (formerly Propionibacterium acnes) strains were isolated at the laboratory of bacteriology of Reims University hospital (CHU Reims). Strains were identified by mass spectrophotometry in routine by University hospital. Four clinical isolates were non-infection related strains (C2, C5, C8, and C18) and after being anonymized, these strains were labeled as infection-naive strains. Nine other C. acnes strains were isolated from bone and prosthesis infections BPIs (BPI 1 to BPI 9). They were defined when at least three of the five samples from the bone and joint tissue during orthopedic surgery were positive. C. acnes strains were isolated on Columbia agar with 5% sheep blood (BioRad, Hercules, CA, USA) and cultivated in a Brain Heart Infusion (BHI) broth (BioRad, Hercules, CA, USA) for five days under anaerobic conditions using the GenBox system (Biomerieux, Marcy l’Etoile, France) at 37 °C.
3
Microbiological Evaluation of Samples
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Microbiological evaluation was performed following the protocols established by the Applied and Environmental Microbiology Laboratory of Animal Health and Anatomy Department of the Veterinary Faculty (UAB). Samples were grown in blood agar (Columbia agar supplemented with sheep blood), Man–Rogosa–Sharpe agar (MRS, tryptic soy agar (TSA), McConkey agar (MK), Baird–Parker agar (BP) supplemented with egg yolk tellurite emulsion, Sabouraud agar supplemented with 0.5 gr/L chloramphenicol, and tryptone sulfite neomycin (TSN) agar. All culture media were purchased from Liofilchem Srl (Italy), with the exception of Columbia agar, which was purchased from Bio-Rad Laboratories (USA). Oxygen and temperature conditions for the different culture media are shown in Table 1 .
Samples were processed within one hour of sampling. Swabs were streaked on Petri dishes following a continuous streak method. Then, plates were incubated for 24 h at the different stated conditions and an initial counting of colonies was performed. Those plates with negative growth were incubated during 24 more hours and the counting was repeated at the end of this second period of incubation. Plates in anaerobic conditions were incubated for 48 h with an Anaerocult system (Merck KGaA, 64271 Darmstadt, Germany), whereas Sabouraud agar plates were incubated for 7 days before being discarded.
Samples were processed within one hour of sampling. Swabs were streaked on Petri dishes following a continuous streak method. Then, plates were incubated for 24 h at the different stated conditions and an initial counting of colonies was performed. Those plates with negative growth were incubated during 24 more hours and the counting was repeated at the end of this second period of incubation. Plates in anaerobic conditions were incubated for 48 h with an Anaerocult system (Merck KGaA, 64271 Darmstadt, Germany), whereas Sabouraud agar plates were incubated for 7 days before being discarded.
4
Whole Genome Sequencing of Bacterial Strains
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The collection of strains that was subjected to WGS include not only strains of the main serotypes found in clinical human or animal infections but also less frequent serotypes (serotypes 5, 10, 11, 12, 14, 16, 18, 27, 29) (Table S1).
Bacterial strains (n=102), stored at −80 °C in buffered peptone water solution with glycerol (20 %), were sub-cultured on Columbia Agar with 5 % Sheep Blood (Bio-Rad) and incubated overnight at 37 °C under 5 % CO2.
Bacterial strains (n=102), stored at −80 °C in buffered peptone water solution with glycerol (20 %), were sub-cultured on Columbia Agar with 5 % Sheep Blood (Bio-Rad) and incubated overnight at 37 °C under 5 % CO2.
5
Bartonella Blood Culture Protocol
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Blood was cultured for Bartonella spp. in accordance with the Centers for Disease Control and Prevention (CDC) procedures (Ying et al. 2002 (link), Bai et al. 2009 (link)). Briefly, whole blood was diluted 1:4 in brain heart infusion media containing 5% amphotericin B (Sigma) to reduce the likelihood of fungal contaminants because of the slow growth of Bartonella spp. Diluted blood samples were pipetted onto Columbia agar (BioRad) plates containing 5% fresh sheep blood previously screened to ensure absence of Bartonella spp. Plates were incubated at 35°C in an atmosphere of 5% CO2 for up to 4 weeks. Plates were examined once per week for bacterial growth. Bacterial colonies were selected on the basis of colony morphology, size and shape, and Gram-staining characteristics, and were subcultured from initial plates and subsequent passages until a pure culture was obtained.
6
Microbial Analysis of Raw Milk
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Raw milk samples were examined microbiologically by plating 10 μL of each sample on the following media: Columbia agar with 5% blood; and Columbia CNA Agar with 5% blood, drigalski agar, and mannitol salt agar (all from BioRad, Marnes-la-Coquette, France). The plates were incubated at 37 °C for 24–48 h. Blood agar plates were incubated in 5% CO2. The culture was considered positive if there was a growth of individual bacteria in a concentration of more than 104 CFU/mL. All isolates were identified and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using a VITEK MS instrument (bioMérieux, Marcy l’Etoile, France).
7
Isolation of Lactobacilli from Infant Stool
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Stool samples were collected from healthy babies aged less than 4 months. Fecal samples were collected either from the diaper or by rectal swabbing (n=72). After serial dilution, samples were cultured on the Man, Rogosa and Sharpe agar medium (MRS, Bio-Rad, France). The culture was incubated for 24 or 48 hours at 35°C in anaerobic conditions by using anaerobic bags (Biomérieux, France).
In order to avoid the growth of unwanted species, in particular enterococci, the subculture of suspected colonies was carried out using a selective medium of the following composition: Columbia agar (Bio-Rad, France) supplemented with glucose, lactulose, cysteine HCL, riboflavin (Sigma-Aldrich, Germany), propionic acid and Mupirocin (GlaxoSmithKline, France) as an antibiotic. This medium is specific for the growth of lactobacilli and other LAB strains, and subsequently inhibits the growth of enterococci and yeasts (16 (link)). All mothers accepted to participate in this study and signed informed consent prior sample collection.
In order to avoid the growth of unwanted species, in particular enterococci, the subculture of suspected colonies was carried out using a selective medium of the following composition: Columbia agar (Bio-Rad, France) supplemented with glucose, lactulose, cysteine HCL, riboflavin (Sigma-Aldrich, Germany), propionic acid and Mupirocin (GlaxoSmithKline, France) as an antibiotic. This medium is specific for the growth of lactobacilli and other LAB strains, and subsequently inhibits the growth of enterococci and yeasts (16 (link)). All mothers accepted to participate in this study and signed informed consent prior sample collection.
8
Identification of Campylobacter Strains
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Once purified onto Columbia agar (Bio Rad) with 5% horse blood (IPA), identification of Campylobacter strains was performed using the standard tests: Gram stain, motility, catalase, and oxidase reactions, growth at 25°C and aerobic growth. Suspected colonies of Campylobacter were subjected to confirmation by studying biochemical tests on triple sugar iron agar (IPA) and by testing their sensitivity to nalidixic acid (NA) (30 µg) and cephalothin (KF) (30 µg) [11 ,15 ]. After that, only one strain from each Campylobacter-positive sample was selected for susceptibility testing.
9
Identification of E. coli Hemolysis Activity
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Columbia agar supplemented with 5% (v/v) sheep blood (Bio-Rad, Marnes-la-Coquette, France) were used to identify haemolysis activity in the E. coli isolates. Indication of alpha-haemolysin and all haemolysin types (beta and gamma) were examined after 6 h and 24 h respectively (Schmidt & Karch, 1996) . To test for the presence of hlyA gene, PCR was then performed on the isolates which had given positive results for alpha-haemolysis activity (Lorenz et al., 2013) . The 20 μL reaction mixture consisted of 1 μL (100ng) DNA template, 2 μL (10 μM) of each hlyA primers (Table 1), 10 μL of 2 times KAPA Taq ReadyMix PCR Kit and PCR grade water.
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