Study population and study design
This is a prospective interventional cohort study of healthy S. aureus carriers and noncarriers in the Netherlands. All experiments were performed in accordance with the Dutch Medical Research Involving Human Subjects Act (WMO). The study protocol was approved by the local Medical Ethical Committee of the Erasmus University Medical Centre Rotterdam, The Netherlands (MEC-2018-091). Written informed consent was obtained for all participants. Participants were recruited through advertisements at Dutch universities and the research teams social networks. Exclusion criteria were age < 18 years, use of antibiotics, antiparasitics, antifungals or probiotics 3 months prior to recruitment, known allergy to components of the intervention treatment, pregnant and breastfeeding women, known chronic diseases affecting the immune system, severe chronic skin diseases, immunocompromised status or use of immunosuppressant drugs.
After filling out an eligibility questionnaire, all volunteers were screened for S. aureus carriage as described previously 21. S. aureus carriage was determined by quantitative culture of 2 weekly nasal swabs. Persistent S. aureus carriers were defined as 2 positive cultures with > 8 CFU for each culture. Noncarriers were defined as 2 S. aureus-negative cultures. Intermittent S. aureus carriers were excluded from further participation in the study. Eligible volunteers were enrolled on a first-come, first-served basis.
Eligible participants were asked to fill out a questionnaire regarding risk factors for S. aureus acquisition. All participants received decolonization treatment. Decolonization consisted of mupirocin nasal ointment (2%, GlaxoSmithKline BV, Zeist, the Netherlands) twice daily and chlorhexidine gluconate cutaneous solution (4%w/v, Regent Medical Overseas Limited, Oldham, UK) once daily, both for 5 days.
Nasal samples were taken 1 day before decolonization (D0) and 2 days (D7), 1 month (M1), 3 months (M3) and 6 months (M6) after decolonization. All participants received a personal demonstration for nasal sampling by the executive researcher. Thereafter, all specimen were taken by the participants by inserting a swab (ESwab, 490CE.A, Copan Italia, Brescia, Italy) into one nostril and rotating 5 times, repeating this in the second nostril using the same swab. Swabs were collected in a container filled with 1 ml modified Liquid Amies, a collection and transport solution, and sent through regular mail service (non-temperature controlled) or deposited at the laboratory personally.
S. aureus quantitative culture
Quantitative S. aureus cultures were conducted to examine the dynamics of S. aureus carriage over the 6-month follow-up period after decolonization. Swab containers were vortexed for 20s before plating. Serial dilutions of Amies medium were plated onto phenol mannitol salt agar (PHMA) and incubated for 2 days at 37°C. Swabs were placed in phenol mannitol salt broth (PHMB) and incubated for 7 days at 37°C for enrichment. S. aureus growth confirmed by a latex agglutination test (Staph Plus Latex Kit, Diamondial, Vienna, Austria). Morphologically different S. aureus colonies were selected for spa typing and methicillin resistance screening using BBL CHROMagar MRSA II agar (BD, Breda, the Netherlands).
Molecular typing of S. aureus isolates was performed to infer whether recolonization with S. aureus in decolonized carriers involved the same spa-type. S. aureus DNA lysates were prepared by boiling in 10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0 or extraction with the QIAamp DNA Mini Kit (QIAGEN, Venlo, The Netherlands) according to the manufacturer's instructions. Amplification of the S. aureus protein A (spa) repeat region was performed by PCR with 2 sets of primers. One set consisted of forward primer spa-1113, 5'-TAAAGACGATCCTTCGGTGAGC-3' and reverse primer spa-1514, 5'-CAGCAGTAGTGCCGTTTGCTT-3' 22. The other set consisted of forward primers spa-F1, 5’- AACAACGTAACGGCTTCATCC-3’ and spa-F2 5’- AGACGATCCTTCAGTGAGC-3’and reverse primer spa-R1 5’-GCTTTTGCAATGTCATTTACTG-3’. Amplicons were purified with ExoSAP-IT (Applied Biosystems) according to the manufacturer’s instructions and sent for sequence analysis (Baseclear, Leiden, the Netherlands). Resulting sequences were analysed using BioNumerics v7.6 (Applied Maths NV, Sint-Martens-Latem, Belgium) and the spa types were assigned by use of the RidomStaphType database (Ridom GmbH, Würzburg, Germany).
16s Ribosomal Rna Sequencing Of Nasal Microbiota
The impact of decolonization on the nasal microbiome and the recovery of the microbiome structure after decolonization were examined by means of 16S rRNA metabarcoding. Amies medium from each nasal swab container was stored at -80°C on the day of receipt at the study laboratory in Rotterdam, NL, then sent at -80°C to the microbiome analysis laboratory in Lyon, FR. To properly capture the impact of decolonization on the living microbiota, metabarcoding used RNA-based 16S ribosomal RNA (rRNA, which is preserved in living cells but quickly cleared after cell death or lysis) rather than the DNA coding sequence, as DNA can persist for prolonged time periods after cell death 23–26. RNA was extracted using the Mag Bind® Total RNA 96 Kit (Omega Bio-tek) tissue protocol from 150 µL of samples’ material. Cell lysis was performed using beads (Disruptor plate C plus – Omega Bio-tek) and proteinase K for 15 min at 2600 rpm, followed by 10 min at room temperature without agitation, and finished with a DNase I digestion of 20 min at room temperature. RNA was quantified using QuantiFluor RNA kit on Tecan Safire (TECAN). 10 ng total RNA was used for reverse transcription using FIREScript RT cDNA synthesis kit (Solis Biodyne) with random primers, then cDNA was purified with SPRIselect reagent (Beckman coulter) and quantified.
The rRNA V1-V3 region was PCR amplified using the 5X HOT BIOAmp ® BlendMaster Mix 12,5 mM MgCl 2 (Biofidal), 10X GC rich Enhancer (Biofidal) and BSA 20 mg/ml. The PCR reaction consisted of 30 cycles at 56°C using the forward primer 27F, 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG AGAGTTTGATCCTGGCTCAG-3’ and reverse primer 534R, 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATTACCGCGGCTGCTGG-3’ in 25 µL of solution. PCR products were purified using SPRIselect beads (Beckman Coulter) in 20 µL nuclease-free water and quantified using QuantiFluor dsDNA (Promega). Samples were indexed with lllumina’s barcodes with the same PCR reagents during a 12 cycles PCR, then purified and quantified as previously mentioned. Samples were normalized and pooled, then sequenced using Illumina MiSeq V3 Flow Cell following the constructor’s recommendations for a 2x300 bp paired-end application. A mean of 130k proofread reads per sample was obtained.
Experiment buffers were used as negative controls to detect contamination by out-of-sample bacterial RNA. RNA extraction was controlled using an in-house mix of live Staphylococcus aureus ATCC29213 and Escherichia coli ATCC25922 in equal proportions, allowing for assessing extraction bias in Gram-positive and -negative bacteria. PCR amplification bias was controlled using a commercial DNA mix of 8 bacterial species (ZymoBIOMICS™ Microbial Community DNA Standard).
Bioinformatics And Statistical Analyses
Sequencing reads were quality checked and trimmed. Paired-ended read pairs were merged using BBMap version 38.49 (available at https://sourceforge.net/projects/bbmap/), with default options besides a minimum single size of 150bp with an average Phred quality score higher than 10, and a total pair size of minimum 400bp. PCR adapters were removed with cutadapt v.2.1 (Martin 2011) then dereplicated using vsearch v.2.12.0 27 with the sizeout option. For species assignment, reads were aligned to sequences of NCBI blast 16S_ribosomal_RNA database (version date 03.12.2020) using Blastn v.2.11.0 + 28,29, keeping a maximum of 20 reference targets. Read counts per bacterial species were normalized to account for taxon-specific variations of the copy number of 16S rRNA genes using NCBI rrnDB-5.5 database based on the mean gene copy number in the taxon.
To optimize the resolution of sequencing read taxonomic assignment, we used in-house bioinformatic software publicly available at https://github.com/rasigadelab/taxonresolve. Briefly, when a read matches sequences from several species with identical alignment scores, taxonomic assignment pipelines typically output the higher taxonomic level such as the genus (e.g., Staphylococcus spp. when a read matches S. aureus and S. epidermidis). This loss of information can be problematic when species-level discrimination is important. To prevent losing species-level information, the taxonresolve software assigns reads with uncertain species to groups of species rather than to genera.
Bacterial species deemed present from contaminating sources such as kits reagents and found in negative controls, mostly from the Bacillus genera, were removed. A total of 1,376 species or group of species were retained. The rarefaction curves corresponding to the sequencing effort to assess the species richness within samples are shown in Supplementary Fig. 3. Most samples reached a plateau after 40 000 sequences.
Given the small sample size compared to the number of variables and species considered in this study, no hypothesis testing was performed and we provide a descriptive assessment of the results. In figures, 95% confidence intervals of the means were computed based on normal approximation, after log transformation for CFUs and log odds transformation for quantities restricted to the [0,1] interval, such as proportions.
In microbial diversity analyses, we retained the 9 most prevalent bacterial species and pooled the other species into an ‘Others’ category. To assess the disruption and possible recovery of the microbiota, the divergence of sampled microbiota relative to the initial, pre-treatment microbiota (D0) was assessed using the Bray-Curtis dissimilarity at each sampling time point relative to the first sample of the same patient.
Software code of the analyses are available at https://github.com/rasigadelab/macotra-metabarcoding. Data are available at https://zenodo.org/record/6382657. Analyses and figures used R software v3.6.0 30 with packages dplyr 31, ggplot2 32, vegan 33, and MicrobiomAnalyst available at https://www.microbiomeanalyst.ca 34,35.