Flow Cytometric and 16S Sequencing Methodologies for Monitoring the Physiological Status of the Microbiome in Powdered Infant Formula Production

The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility.


Supplementary Data 1: Fluorophores' stock and working solutions.
Fluorophores SYTO62: As it was mentioned above, SYTO62 cell-permeant nucleic acid stain was used for discriminating between the cells of interest and the debris. Working solution of SYTO62 (5 µM) was prepared by first warming a vial of the stock solution (5 mM; Molecular Probes, S11344, USA) and bringing it to room temperature. It was then briefly centrifuged (short-spin of 2-3 s) in order to deposit the dimethyl sulphoxide (DMSO) which could interfere with the staining of the cells. Of the stock solution, 10 µL was added to 9,990 µL of filter-sterilized Tris-HCl-EDTA solution (50 mM Tris-HCl and 1 mM EDTA). The latter was prepared by dissolving 6.057 g of Trizma ® base (Tris; Sigma-Aldrich 93349) in 800 mL of deH2O followed by addition of 10 mL of 100 mM Ethylenediaminetetraacetic acid (EDTA). The pH of the solution was then adjusted to 7.5 with 1 M HCl (88.33 mL/L of 35% HCl; VWR 20246.298, Ireland). Finally the volume was adjusted to 1 L. The working solution of SYTO62 was divided into aliquots of 1 mL and stored at -18 °C until use. EDTA is a chelating agent and a scavenger of metal ions. It facilitates the permeabilisation of the outer membrane of the cells, particularly the Gram negative cells by removing the excess extracellular Mg 2+ and Ca 2+ as well as reducing the interaction between the lipopolysaccharide (LPS) molecules of the membrane. EDTA solution (100 mM) was prepared by dissolving 14.612 g of EDTA (Sigma ED, USA) in 400 mL of deH2O with vigorous mixing, adjusting the pH with 10 N NaOH (10 M) to 8.0 and adjusting the volume to 500 mL. It was then filter sterilized to remove the particulates and autoclaved at 121 °C for 15 min.
PI: The cell-impermeant PI is the most commonly used dye for determining membrane integrity, hence viability of the cells. It binds to the DNA of cells that have lost their membrane integrity (dead cells) and is generally excluded from cells with an intact membrane (viable). The working solution of PI was prepared by dissolving 4 mg of PI powder (Sigma P4170; ) in 20 mL of deH2O (299.23 µM). The solution was filter-sterilized using 0.22 µm syringe filters, divided into 1 mL aliquots and stored at 4 °C until use.
BOX: The potentiometric anionic dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)], also known as bis-oxonol or BOX was used in this study for detecting cells with depolarized membrane (i.e. injured or dead). In this study, BOX was used in combination with PI (i.e. triple-staining with BOX, PI and SYTO62) in order to identify three distinct subpopulations of healthy (PI − /BOX − negative; intact membrane integrity and membrane potential), injured (PI − /BOX + ; intact membrane but collapsed membrane potential) and dead (PI + /BOX + ; compromised membrane and collapsed membrane potential) cells by plotting green and red fluorescence parameters against each other. BOX stock solution (19.36 mM) was prepared by dissolving 25 mg of BOX (Sigma D8189; USA) in 2.5 mL DMSO (Sigma D8418; USA). It was then divided into 100 μL aliquots and stored at -20 °C until use. Stock solutions were not filter-sterilized. In order to prepare the working solution (19.36 μM), the stock solution was defrosted, 5 μL was added to 4,795 μL of filter-sterilized PBS and supplemented with 200 μL of filter-sterilized 100 mM EDTA (described above). Working solutions were stored for up to a week at 4 °C or at -20 °C until use.
TO and SYTO9: In order to further investigate the viability status of the cells, samples were also dual-stained with PI and either of SYTO9 or its parent compound thiazole orange (TO). Cellpermeable green fluorescent dyes of SYTO9 and TO stain all cells regardless of their physiological state, to varying degrees. However, when used with PI the competition between PI with SYTO9 or TO for DNA binding sites in dead cells and the subsequent displacement of the latters with PI reduces the green fluorescence intensity of the dead cells. Consequently, this leads to identification of two distinct fluorescent subpopulations of green (TO + /PI − or SYTO9 + /PI − ; viable) and red (TO − /PI + or SYTO9 − /PI + ; dead) when the green and red fluorescent parameters are plotted against each other. Working solution of TO (42 µM) was obtained from BD Biosciences (Cell Viability Kit 349483, BD Biosciences; Oxford, UK). Working solution of SYTO9 (250 µM) was prepared by first, warming the vial of stock solution (5 mM; Molecular Probes, S-34854) to room temperature. Stock solution (40 µL) was added to 760 µL DMSO, vortexed and stored at -20 °C for up to a year. SYTOX Green Dead Stain: The cell-impermeant nucleic acid stain of SYTOX Green Dead Cell Stain (Molecular Probes, S-34860) was used as an alternative viability dye to propidium iodide in order to determine the membrane integrity of the cells. SYTOX stock solution (30 µM) was thawed and allowed to equilibrate to room temperature, after which it was diluted in a 1:10 ratio in DMSO (3 µM). The working solutions were divided it ten aliquots of 100 µL (in 0.2 mL microcentrifuge tubes) and stored at -20 °C until use.
Hexidium iodide (HI): HI was used in combination with SYTO9 in order to determine the Gram characteristics of the cells. When bound the to the DNA, the maxima excitation/emission wavelengths for SYTO9 and HI are 485⁄498 nm and 518/600 nm, respectively. Therefore, while both can be excited by the green laser, the emissions of SYTO9 and HI are collected by FL1 and FL3 detectors, respectively. SYTO9 stains most Gram-negative and Gram-positive bacteria whereas HI preferentially stains the Gram-positive cells. When used together, HI displaces the SYTO 9 stain resulting in a decrease in the green fluorescence of Gram-positive cells. Therefore, when green (FL1) and red (FL3) fluorescence are plotted against each other, Gram positive and negative cells could be identified as red and green fluorescent populations, respectively. In order to prepare the stock solution of HI (10.05 mM), 5 mg of HI powder (Molecular Probes H7593, USA) was dissolved in 1 mL DMSO, divided into 200 µL aliquots and stored at -20 °C until use. For preparing the working solution (25.13 µM). On the day of experiment, the working solution was prepared by diluting the stock solution in PBS in a 1:400 ratio and storing at 4 °C until use.
CTC: CTC was used for studying the respiratory activity of the cells. CTC at its oxidized form is a soluble non fluorescent compound, however, upon its reduction by the electron transfer chain of actively respiring cells, it is converted into non-soluble crystals of red-fluorescent CTCformazan. The rate of intracellular accumulation of CTC-formazan could be used as a semiquantitative indicator of the number of healthy and non-healthy cells within the microbial population. Working solution (53.46 mM) of CTC was prepared on the day of experiment by dissolving 5 mg of CTC in 300 µL of ultrapure 0.1 µm filtered water (Sigma, W4502; USA) and stored at 4 °C until use.

Staining buffer
Immediately prior to staining, 250 µL of diluted sample (cell suspension in PBS) was transferred to 12×25 mm flow tubes and supplemented with 20 µL of filter-sterilized 100 mM EDTA and 20 µL of 0.1% (v/v in deH2O) Polyoxyethylene sorbitan monolaurate (Tween ® 20) (Sigma P1379, USA). Therefore, the total volume of an unstained sample (with no added fluorophore) was 290 µL containing 6.90 mM EDTA and 0.007% (v/v) Tween ® 20. Tween ® 20 was used as a mild non-ionic surfactant for improved permeabilisation of the cell membranes. In order to prepare the 0.1 % (v/v) Tween ® 20 solution, 0.1 mL of Tween ® 20 was added to 95 mL of deH2O and the volume adjusted to 100 mL. The solution was then passed through 0.22 µm syringe-filters and stored at 4 °C for up to a week.

Supplementary Data 2: Gating strategy
In summary, the information from P1 was passed through two plots, one representing FSC versus FL5 (plot a[2] and b[2]) and another one SSC versus FL5 (plot a[3] and b [3]). The ranges of FSC and SSC values in plots a(2)/b(2) and a(3)/b(3), respectively, were similar to those in the first plot for the P1 population. By comparing the contour plots of unstained and SYTO 62-stained cells (contour maps of 99% probability), the SYTO 62 positive cells in plots b(2) and b(3) were gated and defined as P2 and P3, respectively. The events present in both P2 and P3 were then passed through two plots, one representing FSC versus FL6 (plots a[4] and b [4]) and the other one SSC versus FL6 (plot a [5] and b[5]) by using the "AND" Boolean logical operator, (i.e. P2 AND P3). FL6 positive populations in plots b(4) and b(5) were gated and defined as P4 and P5, respectively. Using the same Boolean logic, events present in both P4 and P5 gates (i.e. P4 AND P5) of plots b(4) and b(5) were presumed as cells [minus those shown in gate P6 of plot a[6]), plotted in a separate FSC versus SSC-A plot and designated as gate P6. Therefore, any events shown in gate P6 (presumed cells), was also detected in gates P1, P2, P3, P4 and P5. In order to investigate the physiological status of the cells, depending on the number and the type of fluorophores used, the events within P6 were passed through a plot representing either FL1, FL3 or both (FL1 versus FL3).  NA 4.51% -Notes: Dash sign "-": Correction not required; NA: Not applicable; NC: Not Corrected. The spillover of the fluorescence of SYTO 62 on FL6 was not corrected in order to utilize this overspill for gating and differentiating between the cells and the debris as previously described. The highlighted cell indicates the primary detector for that fluorophore. (1) When used in combination with SYTO ® 9 (PMT voltage of 450 V); (2) When used in combination with other fluorochromes (PMT voltage of 600 nm).