Enumerating Virus-Like Particles and Bacterial Populations in the Sinuses of Chronic Rhinosinusitis Patients Using Flow Cytometry

Abstract

There is increasing evidence to suggest that the sinus microbiome plays a role in the pathogenesis of chronic rhinosinusitis (CRS). However, the concentration of these microorganisms within the sinuses is still unknown. We show that flow cytometry can be used to enumerate bacteria and virus-like particles (VLPs) in sinus flush samples of CRS patients. This was achieved through trialling 5 sample preparation techniques for flow cytometry. We found high concentrations of bacteria and VLPs in these samples. Untreated samples produced the highest average bacterial and VLP counts with 3.3 ± 0.74 x 107 bacteria ml-1 and 2.4 ± 1.23 x 109 VLP ml-1 of sinus flush (n = 9). These counts were significantly higher than most of the treated samples (p < 0.05). Results showed 103 and 104 times inter-patient variation for bacteria and VLP concentrations. This wide variation suggests that diagnosis and treatment need to be personalised and that utilising flow cytometry is useful and efficient for this. This study is the first to enumerate bacterial and VLP populations in the maxillary sinus of CRS patients. The relevance of enumeration is that with increasing antimicrobial resistance, antibiotics are becoming less effective at treating bacterial infections of the sinuses, so alternative therapies are needed. Phage therapy has been proposed as one such alternative, but for dosing, the abundance of bacteria is required. Knowledge of whether phages are normally present in the sinuses will assist in gauging the safety of applying phage therapy to sinuses. Our finding, that large numbers of VLP are frequently present in sinuses, indicates that phage therapy may represent a minimally disruptive intervention towards the nasal microbiome. We propose that flow cytometry can be used as a tool to assess microbial biomass dynamics in sinuses and other anatomical locations where infection can cause disease.

International consensus statement on allergy and rhinology: rhinosinusitis 2021

“…In addition to bacterial alterations seen in the microbiome in CRS, viral and fungal changes may also be seen 753–759 . Further in vivo studies of the relationship of viruses and fungi to the sinus microbiome in health, CRS, or AECRS are an area of ongoing interest and will likely evolve with the application of new technologies.…”

The microbial abundance dynamics of the paediatric oral cavity before and after sleep

“…Eluted swab samples were diluted (1:100) in 0.2 µm filtered TE buffer for optimal visualisation of bacterial and virus-like particle (VLP) populations. Diluted samples were then stained with SYBR-I Green (1:20,000 final dilution; Molecular Probes) and incubated for 10 minutes in the dark at 80°C as per previously established and optimised methods [17,18,21]. Control samples of sterile rayon swabs eluted in sterile TE buffer were prepared in the same manner as the participant swab samples.…”

“…Flow cytometry is an inexpensive method used to enumerate and monitor absolute microbial abundance dynamics within numerous environments [16][17][18][19][20][21][22][23]. This technique has been optimised to count microorganisms, including viruses, within samples that would otherwise be deemed too low for epifluorescence and transmission electron microscopy [16,17,23].…”