Bacterial bioload and community structure of drinking water used on the International Space Station

Taking water samples for people with disabilities on the International Space Station

On Jan. 8, 2021, an astronaut collected 350ml of safe water for people with disabilities on the United States-orbited portion (USOS) of the International Space Station in a NASA post-flight analysis bag. It is made of Fluorinated Ethylene Propylene (FEP) with Luer Lock Ports Female Polypropylene and is used for water sample collection and analysis by NASA. A potable water sampling bag from the International Space Station is loaded onto the SpaceX CRS-21 (SpX-21) cargo capsule, which plunged into the Gulf of Mexico on January 14, 2021 and flown to NASA’s Kennedy Space Center (KSC) at room temperature. . . After arriving at KSC, the bag was stored at 4°C and transported from KSC to Tsukuba Space Center, JAXA, Japan, arriving on January 21, 2021. Ground control water was prepared in our laboratory by collecting high-purity water in the same bag at the same time. Water samples for PWD were taken and stored in the same conditions until measurement.

Total direct count and CFU count

Bacteria in the PWD water were filtered through a black polycarbonate filter (0.2 μm pore size, ADVANTEC). The filters were rinsed twice with bacteria-free distilled water. Then, 1 μg/mL DAPI in distilled water or 150 μg/mL 6-CFDA in phosphate buffer (0.3 M phosphate (pH 8.5), 15% NaCl, 1.5 mM EDTA) was applied to the filters and incubated for 2 minutes. 3 minutes at room temperature under dark conditions. Filters were rinsed twice, mounted on glass microscope slides with non-fluorescent oil immersion, and examined with an epifluorescence microscope (DM2500, Leica Microsystems) with an oil immersion objective. CFU number was determined by spreading ISS diluted drinking water onto BD TSA and BD BBL R2A agar (Becton, Dickinson and Company), which were incubated for 1 week before counting at 30 °C and 22 °C, respectively.

MALDI-TOF MS spectrometer for identification of isolated bacteria

MALDI-TOF MS was used for direct identification of bacteria grown on TSA and R2A . agar24. 18 and 20 isolated colonies of TSA and R2A were stained on a polished MALDI MSP 96 steel target plate. One microliter of 70% formic acid was deposited onto each sample spot and left to dry. Then, 1 μl of matrix solution (α-cyano-4-hydroxycinnamic acid (Bruker Daltonics) dissolved in 50% acetonitrile, 47.5% water, and 2.5% trifluoroacetic acid) was applied to each sample spot and allowed to dry. Brucker’s Bacterial Test Standard, Escherichia coli DH5α was used for calibration. Measurements were made with a Microflex mass spectrometer (Bruker Daltonics) using flexControl software version 3.4. Mass spectra were obtained at a linear positive extraction mode ranging from 2,000 to 20,000 Da. Spectra were analyzed using MALDI Biotyper 3.1 software with Bruker BDAL Ver library. 6 The Library of Filamentous Fungi Ver. 1.0 (Broker Daltonix). The cut-off score recommended by the manufacturer was used for identification (Supplementary Table 2).

Counting bacterial cells using a bioparticle counter

A commercially available bio-fluorescent particle counter (XL-10BT1, Rion Co. Ltd.) was used in this study. He had two detectors. One was a photodiode that measured the intensity of scattered light, indicating particle size. The other was a photomultiplier tube to measure the intensity of autofluorescence, an indicator of the physiological activity of the bacteria. This allowed the scattered light intensity and autofluorescence intensity to be measured simultaneously.

We have developed a protocol to measure bacterial cells without staining using a biofluorescent particle counter, identifying autofluorescent particles from flavin and counting them as bacterial cells. Flavin is a ubiquitous pigment in bacterial cells, which emits autofluorescence at 510 nm when irradiated with 405 nm excitation light.25. The amount of intracellular flavin is closely related to the physiological activity of bacteria25, thus bacteria with low physiological activity may not be detected due to the low autofluorescence intensity. To solve this problem, we irradiated the particles with deep UV irradiation, at a wavelength of 254 nm or two wavelengths of 185 nm and 254 nm, to oxidize flavin and enhance the autofluorescence intensity immediately before measurement under irradiation at 405 nm, which was carried out with a deep UV irradiator Ultraviolet (XL-28A, RION Co. Ltd.) is fitted with a low pressure mercury lamp. Since it is known that deep UV radiation degrades organic carbon26, 27, the deep UV irradiation included in the bacterial cell counting protocol was also used to reduce signals from dissolved organic carbon in PWD water. Particle counting was performed at a flow rate of 10 ml/min and a measurement time of 60 s. The deep UV dose was more than 1000 mJ/cm2. Prior to the measurement of bacterial cells, ultrapure water was used to set a cut-off limit for the electrical noise derived from the biofluorescent particle counter, which was defined as 133 mV. To exclude particles with very high autofluorescence density from being considered bacteria, the upper threshold was set at 1200 mV when counting bacterial cells.

DNA extraction

Bacterial cells in 50 ml of PWD water were confined to a sterile polycarbonate membrane filter (0.2 μm pore size; Advantec, Tokyo, Japan). Bacterial DNA was extracted by the method described in Ichijo et al.2. DNA was finally eluted with 50 μl of TE solution.

Amplicon sequencing of the bacterial 16S rRNA gene by the ONT MinION . platform

Amplicon sequencing targeting the full length of the 16S rRNA gene was performed using MinION equipped with an R9.4.1 flow cell (Oxford Nanopore Technologies, Oxford, UK). A 16S rRNA sequence library was generated from 10 μl of extracted DNA using a 16S ribbon coding kit (Oxford Nanopore Technologies). Library generation was performed according to the manufacturer’s instructions except that DNA amplification was performed using a KAPA HiFi HotStart ReadyMix (KAPA Biosystems, MA, USA) with the following thermal cycling conditions: 2 min at 95 °C, 25 cycles of 20 sec at 98 °C, 30 seconds at 60 °C, 2 minutes at 72 °C, and 5 minutes at 72 °C. Sequencing was performed using Oxford Nanopore’s MinKNOW software and base calls were made with Guppy (version 4.3.4) in fast mode using the dna_r9.4.1_450bps_fast.cfg configuration file. FASTQ files also generated for taxonomic classification were analyzed using the cloud-based EPI2ME FASTQ 16S workflow with a quality score of ≥ 7 for quality filtering.

Amplicon sequencing of the bacterial 16S rRNA gene by Illumina MiSeq and iSeq platforms.

Amplicon sequencing targeting the 16S rRNA gene region V4 was performed using a 300 bp MiSeq bipartite platform with MiSeq Reagent Kit v2 and 150 bp iSeq with the iSeq 100 i1 Reagent platform (Illumina, CA, USA). Two-step PCR was performed to generate two-end libraries. In the first PCR, the V4 region of the prokaryotic 16S rRNA gene was amplified from the DNA sample using primers F515 and R806.28 With Illumina dangling adapters. First PCR reactions were performed in triplicate in a 12 μL reaction volume containing 3 μL of template and 0.6 μM of both forward and reverse primers in 1× KAPA HiFi HotStart ReadyMix (KAPA Biosystems) using a 2-min heat cycle at 95 °C, 35 cycles. From 20 seconds at 98°C, 15 seconds at 60°C, 30 seconds at 72°C, and 5 minutes at 72°C. Triple PCR products were pooled and purified using Agencourt AMPure XP (Beckman Coulter, CA, USA) according to the manufacturer’s instructions. A second PCR (12 cycles) was performed to attach double-pointers and Illumina sequencing adapters to purified first PCR products using Nextera XT Index Kit v2 Set C (Illumina). Finally, the indexed amplicons were purified by electrophoresis using E-Gel SizeSelect II Agarose Gels (Thermo Fisher Scientific, MA, USA). DNA concentrations of indexed amplicons were quantified by a Qubit 4 fluorometer using a dsDNA HS assay kit (Thermo Fisher Scientific) and pooled in equal amounts to build the library. The library was diluted to 1 nM and spiked with 20% PhiX Control v3 (Illumina), then diluted to 50 pixels per minute, loaded into MiSeq and iSeq cartridges, and sequenced according to the manufacturer’s instructions.

FASTQ files generated from MiSeq and iSeq sequencing were analyzed separately using the QIIME 2 pipeline.29 (Version 2021.8). Noise reduction sequencing, paired read merging, and chimera filtering were performed using the DADA2 pairwise qiime dada2 perturbative method.30,31 Plug-in in QIIME 2, where parameters have been modified from default settings for both MiSeq and iSeq data as follows: -p-trim-left-f 19, -p-trim-left-r 20, -p-max-ee- f 1.0, and –p-max-ee-r 1.0. In addition to these modifications, the parameters -p-trunc-len-f and -p-trunc-len-r were adjusted to 263 and 226 for the MiSeq data to truncate the reads forward and reverse in position with an average quality score lower than 30. For the iSeq data , the forward and reverse readings are not truncated. The -p-min-interapap parameter, which controls the minimum overlap required for merging paired reads, has been reduced from default (12) to 5 to enable merging. The resulting non-chimeric sequences, called Amplicon Sequence Variants (ASVs), were mapped to taxonomic groups using the “qiime feature-classifier classify-sklearn”31 Using a naive Bayes classifier pre-trained on Silva 138 99% database32 For region 515F/806R of 16S rRNA (silva-138-99-515-806-nb-classifier.qza) with a confidence limit of 0.985.

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