Thioglycolate broth

In a recently published study from Madagascar, 1548 nasal swabs (Copan Nasal Swab Transsystem^®, Brescia, Italy) were collected from students and health care workers [11 (link)]. Plating on Columbia blood agar (OXOID^®, Vienna, Austria) with subsequent biochemical identification and antibiotic resistance testing to identify MRSA isolates was performed as described by Hogan et al. [11 (link)]. For this integrated study, MRSA selective agar (CHROMagar MRSA, MAST Diagnostica, Reinfeld, Germany) was used on site in addition to the methods as described by Hogan et al. Afterwards, the same swabs were stored at 4 °C for 2–22 weeks until airborne shipment without cooling for second processing to Germany.
A total of 1541 nasal swabs were shipped to the Bernhard Nocht Institute for Tropical Medicine Hamburg, Germany, and were analyzed by non-selective broth enrichment in thioglycolate broth (BD, Heidelberg, Germany) and subsequent culture on MRSA selective agar CHROMagar MRSA (MAST Diagnostica). The remaining 7 swabs were lost, so the samples could not be included in the assessment in Germany. MRSA strains were identified by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS), by antibiotic susceptibility testing and by polymerase chain reaction (PCR) as detailed elsewhere [13 (link)].

In the surgical ward, at least three tissue samples from infected bone and soft tissues were collected during surgical debridement, and then processed for microbiology and histopathology. Tissue was homogenized in 3 ml of brain-heart infusion (BHI) broth for 1 min and inoculated onto aerobic sheep blood agar, chocolate agar, anaerobic blood agar and into thioglycolate broth (BD Diagnostic Systems, Sparks, MD). The time limit for processing samples was 6 hours. Aerobic and anaerobic plates were incubated aerobically at 35° to 37°C in 5 to 7% CO2 for 7 days, and anaerobically at 37°C for 14 days, respectively. Additionally, 0.5 ml of tissue homogenate was inoculated in thioglycolate broth, incubated for 14 days, and the thioglycolate broth was sub-cultured on blood agar plates when cloudy. Colonies of microorganisms growing on the plates were identified, and their susceptibility to antibiotics was tested according to standard microbiologic techniques.

Tissue samples were processed according to the following protocol: after arrival at the Department for Infectious Diseases, Medical Microbiology and Hygiene of Heidelberg University, the tissue was ground using a sterile porcelain mortar, followed by the addition of 1 mL of 0.9% NaCl. This suspension was inoculated onto Columbia 5% sheep blood agar (BD life sciences, Heidelberg, Germany), chocolate agar, MacConkey agar, SCS agar, Schaedler Neo Vanco +5% sheep blood (SNVS) agar (all BioMérieux, Marcy, France), and thioglycolate broth (BD life sciences), and then Gram staining was performed. Samples were incubated under aerobic or anaerobic conditions, as appropriate, for 48 h (aerobic microorganisms) or 72 h (anaerobic microorganisms) at 36°C. If growth on plates was detected, identification of microorganisms was performed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Bruker Daltonics, Bremen, Germany) as described elsewhere.24 (link) Susceptibility testing was performed using VITEK2 (Biomérieux, Nürtingen, Germany) or MIC test strips (Liofilchem, Piane Romano, Italy), respectively, and the results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing clinical breakpoints.

Peritoneal effluent specimens that were collected for clinical indications were cultured at Spectra Laboratories (Rockleigh, NJ) per their standard operating procedures or at New York-Presbyterian Hospital/Weill Cornell Medical Center per their standard operating procedures. Peritoneal effluent specimens which were not collected for clinical indications were cultured at New York-Presbyterian Hospital/Weill Cornell Medical Center using a combination of broth and liquid media. Peritoneal effluent was inoculated into aerobic and anaerobic blood culture vials (Becton, Dickinson, and Company [BD]) and incubated in a continuous automated blood culture instrument (BACTEC FX; BD). Specimens were also plated on tryptic soy agar with 5% sheep blood, MacConkey agar, and chocolate agar, and inoculated into thioglycolate broth (BD and Hardy Diagnostics). These media were incubated at 35 °C in 5% carbon dioxide. Finally, a portion of the fluid was inoculated onto Brucella Blood agar (Anaerobe Systems) and incubated under anaerobic conditions. Identification of microorganisms isolated in culture was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Bruker Daltonics, Inc) and antibiotic susceptibility testing was performed using broth microdilution (Beckman Coulter, Inc).

Frozen isolates were inoculated on selective CHROMagar medium ChromID™ C. difficile (CDIF), (BioMérieux, Durham, NC), and incubated at 37 °C, in a Bactron 300 anaerobic chamber (Sheldon Manufacturing, Cornelius, OR, USA) for 48 h. C. difficile colonies appear asymmetric, black in color, and with an irregular border. Final identification was performed using the Bruker Biotyper system (Bruker Daltonics, Bremen, Germany), which is based on the matrix-assisted laser desorption ionization-time of flight (MALDI TOF) technique [12 (link)].
For the experiments, thioglycolate broth (Becton Dickinson, Heidelberg, Germany) supplemented with L-cysteine 0.5 g/L was inoculated with colonies of each bacterial strain. The broths were incubated overnight at 37 °C, under anaerobic conditions, in a Bactron 300 anaerobic chamber (Sheldon Manufacturing).

Viability following exposure to different STs: Bacteria of each ST were grown overnight in thioglycolate broth (Becton Dickinson) supplemented with L-cysteine 0.5 g/L. Then, a sample was taken from each bacterial suspension for turbidity measurement and adjustment to 0.5 McFarland (equivalent to 1.5 × 10⁸ CFU/mL).
50 μl of bacteria (grown overnight) of each ST at an MOI of 1:50 suspended in thioglycolate fluid was added to each well. Then, the plate was incubated at 37 °C, 90% humidity and 5% CO₂ for 5 min, 10 min, 1 h and 4 h.
Viability following exposure to toxins: 50 μl of toxin A/toxin B/both toxins A + B at different concentrations were added to each well. Then, the plate was incubated at 37 °C, 90% humidity and 5% CO₂ for 30 min, 6 h, 12 h and 24 h. In order to prepare the desirable toxins concentrations, native C. difficile Toxin A and B, (Abcam, Toronto, Canada) were dissolved in DDW and serial diluted in DDW to the following concentrations: 5 ng/μl, 1 ng/μl, 0.5 ng/μ and 0.05 ng/μl.
Following macrophages exposure to the different ST strains or to bacterial toxins, macrophages’ viability was tested using the Cell Proliferation Kit -XTT based (Biological industries, Beit Haemek, Israel); following addition of XTT reagent solution, OD₄₅₀ was measured using an ELISA reader (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Bacteria were grown in thioglycolate broth (Becton Dickinson, USA) for 48 h in anaerobic conditions (85% N₂, 5% H₂, and 10% CO₂) to obtain a cell pellet from 1 mL of culture. DNA was extracted using DNeasy Mini Kit (Qiagen, Hilden Germany) as previously described [15 ,26 (link),27 ]. The quality and yield of genomic DNA was verified by agarose gel electrophoresis and a Nanodrop 2000 (ThermoFischer Scientific, USA). Whole-genome sequencing (WGS) was done using Illumina HiSeq 2000 with PE 150 plus index read (Illumina, San Diego, CA). Adapters and phiX Illumina standards were informatically removed before quality control of the reads was done using FastQC. The coverage was calculated with the formula C = LN/G, where L is the read length, N is the number of reads and G is the genome length [28 ]. The genome sequences were assembled and annotated as previously described [15 ,16 (link),26 (link)]. Briefly, the reads were assembled using Shovill (https://github.com/tseemann/shovill) with the default settings. Automated gene annotation was done with Prokka v.1.14.5 using default parameters [29 ].