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moistened with sterile saline (Copan Diagnostics, Murrieta, CA). Set A included swabs collected from 10 single-use disposable stethoscopes directly from the box prior to use (clean stetho- scopes), 20 single-use disposable stethoscopes in-use in inpatient rooms (patient-room stethoscopes), and 20 stethoscopes being carried by physicians, nurses, and respiratory therapists (practi- tioner stethoscopes). Because low levels of microbial DNA are ubiquitous and bacterial DNA derived from collection instru- ments, reagents or the environment can confound microbiome studies,10,11 a set of background controls comprised of swabs moistened with saline (collected in parallel with the stethoscope sampling) were obtained on each collection date (n=20). Swabs were stored at −80°C.
Standardized cleaning method (set B)
We sampled 10 additional ICU practitioner stethoscopes using the procedure described above, following which stethoscope diaphragms were cleaned with a hydrogen peroxide wipe (hydrogen peroxide 1.4%; Clorox Healthcare, Oakland, CA) for 60 seconds and allowed to dry. Stethoscopes were then swabbed again using the same protocol.
Practitioner preferred cleaning method (set C)
An additional 20 practitioner stethoscopes were collected. To ensure that the pre-cleaning swab would not affect the post- cleaning communities, the stethoscope diaphragm was sampled as described above, but only the left half of the diaphragm was swabbed. Stethoscopes were returned to the practitioner, and they were instructed to clean their stethoscopes using the method they usually would use to clean it between patients. Practitioners cleaned their stethoscopes with hydrogen peroxide wipes (n=14), alcohol swabs (70% isopropyl alcohol; Coviden-Webcol, Mans- field, MA) (n=3), or bleach wipes (sodium hypochlorite 0.55%; Clorox Healthcare) (n=3), and duration of cleaning was deter- mined by practitioner preference. Once the diaphragm was visibly dry, the right half of the diaphragm was then swabbed to capture post-cleaning bacterial communities.
Bacterial extraction
Swabs were cut directly into PowerSoil beadbeater tubes (MoBio, Quagen, Venlo, Netherlands). DNA was extracted using the PowerSoil DNA kit (MoBio), according to the manufacturer’s instructions except for an additional 10-minute, 95°C incubation step to improve DNA yield from hard-to-lyse bacteria. DNA was stored at −20°C.
Bacterial amplification sequencing and analysis
Extracted DNA was amplified in triplicate 25-µL reactions using barcode-labeled primers targeting the bacterial 16S rRNA gene variable regions 1 and 2 (V1V2), employing previously described polymerase chain reaction (PCR) primers and protocols.12,13 The PCR primers target sequences that are conserved among bacteria, whereas the region amplified is not conserved and provides sequence-based information for bacterial identification. Samples were purified using bead purification, quantified using PicoGreen (Fisher Scientific, Waltham, MA), normalized to 5 ng/μL per sample, pooled, and subjected to deep sequencing on the Illumina MiSeq platform. Clean swabs moistened in sterile saline and dry clean swabs were run in parallel as controls for instrument- or
Vincent R. Knecht et al
reagent-derived background sequences. Using the QIIME 1.91 pipeline,14 sequences were clustered based on 97% similarity into de novo operational taxonomic units (OTUs), which serve as the basic taxonomic unit for subsequent analysis. To identify the bacteria, OTUs were aligned to the Greengenes reference database of bacterial sequences. Stethoscope set C was also amplified using primers targeting the 16S rRNA gene V4 region15 and was sequenced and analyzed using the same protocol. Sequences aligning with Streptophyta, which represent chloroplast DNA, were removed from the analysis.16 Bacterial DNA was quantified using 2 methods. First, the
amount of amplification product generated by bar-coded PCR primer amplification during library preparation was used to estimate the relative amount of contamination in pre- and post- cleaning specimens, as previously described.17 In addition, 16S rRNA gene qPCR was carried out on a subset of samples using primers and protocols previously described.18 To identify bacteria (taxa) that are commonly associated
with HAIs, we specifically queried sequences from practitioner stethoscope samples of sets A and C for the presence of Staphy- lococcus, Pseudomonas, Acinetobacter, Clostridium, Enterococcus, Stenotrophomonas, and Burkholderia genera. The 16S rRNA gene sequences assigned to these genera in the QIIME pipeline were then manually aligned to the NCBI 16S rRNA sequence database with BLAST to confirm genus identity and, if possible, to generate species-level assignment. Because any 16S rRNA gene primer set may have unrecognized biases,19,20 we did this using both V1V2 and V4 sequences of the 16S rRNA gene. Any sample with ≥10 sequence reads aligning to the genus was considered a positive hit.
Statistical analysis
Figures were generated and statistical tests carried out using R version 3.2.3 software (R Foundation for Statistical computing, Vienna, Austria). Richness (number of taxa) and alpha diversity (within-community measure that encompasses both richness and evenness) was carried out after sequence rarefaction to 1,000 reads.21 Alpha diversity was calculated using the vegan package. The pairwise Wilcoxon rank-sum test was used to compare between-group differences for the alpha diversity analysis. We analyzed β diversity (a metric of between-community differences) was analyzed by unweighted and weighted UniFrac using the QIIME pipeline, and was used to perform principal coordinate analysis (PCoA).14,22 The adonis function in the vegan package was used to test for statistical significance between groups of communities using PERMANOVA in the β diversity analyses. The Wilcoxon rank-sum test was used to examine the differences between groups of communities in quantification, richness and diversity, and the Student t test was used for the pre- and post- cleaning 16S quantification differences.
Results Stethoscope microbiome community analysis
We first estimated total bacterial contamination of practitioner stethoscopes, patient-room stethoscopes, clean unused stetho- scopes, and background controls in set A according to the quantity of 16S amplicon post-PCR amplification (Fig. 1(A)). Practitioner stethoscopes had significantly higher 16S amplicon concentration compared to both the patient-room and clean stethoscopes (P=.035 and P=.004, respectively; Wilcoxon rank- sum test). Patient-room stethoscopes had significantly higher
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