Alteration of Bacterial Communities in Anterior Nares and Skin Sites of Patients Undergoing Arthroplasty Surgery: Analysis by 16S rRNA and Staphylococcal-Specific tuf Gene Sequencing

Abstract

The aim was to study alterations of bacterial communities in patients undergoing hip or knee arthroplasty to assess the impact of chlorhexidine gluconate soap decolonisation and systemic antibiotic prophylaxis. A Swedish multicentre, prospective collection of samples obtained from elective arthroplasty patients (n = 83) by swabbing anterior nares, skin sites in the groin and the site of planned surgery, before and after arthroplasty surgery, was analysed by 16S rRNA (V3-V4) gene sequencing and a complementary targeted tuf gene sequencing approach to comprehensively characterise alterations in staphylococcal communities. Significant reductions in alpha diversity was detected for both bacterial (p = 0.04) and staphylococcal (p = 0.03) groin communities after arthroplasty surgery with significant reductions in relative Corynebacterium (p = 0.001) abundance and Staphylococcus hominis (p = 0.01) relative staphylococcal abundance. In nares, significant reductions occurred for Staphylococcus hominis (p = 0.02), Staphylococcus haemolyticus (p = 0.02), and Staphylococcus pasteuri (p = 0.003) relative to other staphylococci. Staphylococcus aureus colonised 35% of anterior nares before and 26% after arthroplasty surgery. Staphylococcus epidermidis was the most abundant staphylococcal species at all sampling sites. No bacterial genus or staphylococcal species increased significantly after arthroplasty surgery. Application of a targeted tuf gene sequencing approach provided auxiliary staphylococcal community profiles and allowed species-level characterisation directly from low biomass clinical samples.

Staphylococcal Communities on Skin Are Associated with Atopic Dermatitis and Disease Severity

“…Bacterial cells were enzymatically pre-lysed and DNA extracted using a MagNA Pure 96 purification instrument (Roche, Mannheim, Germany). The V3–V4 region of the 16S rRNA gene was selected for the analysis of complete bacterial communities [ 38 ], and the tuf gene was selected for analysis of staphylococcal species [ 32 ]. The selected gene regions were amplified in a two-step PCR and sequenced on a MiSeq instrument using 600-cycle MiSeq Reagent Kit v3 (Illumina Inc., San Diego, CA, USA) kits.…”

“…DADA2 v.1.12.1 [ 39 ] was used for the inference of amplicon sequence variants (ASVs) and taxonomic assignments using the Silva reference database v.132 [ 40 ] for 16S rRNA-derived ASVs and an inhouse reference database was used for tuf -derived ASVs [ 32 ]. The tuf reference database can be accessed here: , accessed on 18 February 2021.…”

“…Whereas many studies have focused on the role of S. aureus in AD, only a few, and often smaller, studies focusing on CoNS at species level have been published [ 5 , 19 , 30 , 31 ]. One of the reasons for this may be that many studies investigating the skin microbiota are based on targeted amplicon sequencing of 16S rRNA gene regions (V1–V3 or V3–V4 regions) that suffer from limited staphylococcal species discrimination of [ 32 , 33 ].…”

“…Here, our primary aim was to study the diversity of the staphylococcal communities on the lesional and non-lesional skin of AD patients and skin of healthy individuals using an amplicon sequencing-based method targeting the tuf gene. The tuf gene is conserved within the Staphylococcus genus but contains highly variable regions that can be used to discriminate between different species [ 32 ]. We also investigated staphylococcal communities on AD skin in correlation to AD severity and skin barrier dysfunction, including transepidermal water loss (TEWL) and skin pH.…”

Changes in Skin and Nasal Microbiome and Staphylococcal Species Following Treatment of Atopic Dermatitis with Dupilumab

“…The V3-V4 region of the 16S rRNA gene and that of the tuf gene were amplified in two separate PCRs (95 °C for 3 min; 25 cycles of 98 °C for 20 s, 60 °C for 15 s, 72 °C for 45 s; 72 °C for 5 min), using primers (16SrRNA: 341F: 5′- CCTACGGGNGGCWGCAG -3′; 805R: 5′- GACTACHVGGGTATCTAATC-3′; tuf : F: 5′- CAGAAGAAAAAGAACGTGG-3′; R: 5′- GTCCTCAACWGGCATCA-3′) with preceding heterogeneity spacers [ 23 , 24 ]. Amplicon libraries were constructed using nextera indexing primers (Illumina Inc., San Diego, CA, USA) (PCR program used: 95 °C for 3 min; 20 cycles of 98 °C for 20 s, 55 °C for 15 s, and 72 °C for 45 s; 72 °C for 5 min) and sequenced on a MiSeq instrument using a 600 cycle V3 kit (Illumina Inc., San Diego, CA, USA).…”