Non-Culture-Based Analysis of Bacterial Populations from Patients with Chronic Rhinosinusitis

Abstract

Middle meatus aspirates from patients with chronic rhinosinusitis were analyzed by bacterial culture, denaturing gradient gel electrophoresis (DGGE), and antibiotic sensitivity techniques. DGGE detected a greater bacterial diversity than culture methods. Although resistance to antibiotics was low, there was evidence of changes in the composition of the bacterial microbiota over time, and the presence of noncultured bacteria was demonstrated. CASE REPORTUsing a glass canula, aspirates were taken from the middle meatus of six patients who had previously undergone surgery for chronic rhinosinusitis. These patients had continued infection after surgery despite ongoing nasal toilet and antibiotic treatment. The antibiotics prescribed to the patients within the 2 months prior to the aspirates being taken are listed in Table 1. Four of the patients had a second aspirate taken 2 to 3 months after the first sample. The aspirates were first suspended in 2 ml phosphate-buffered saline and then thoroughly homogenized by being vortexed before being divided into two equal volumes for use in culture-based and DNA-based analyses. For the isolation of staphylococci, mannitol salt agar (Difco, Sparks, MD) was incubated at 37°C aerobically. Total bacterial populations were isolated on chocolate agar (Columbia agar base [Difco] with 5% heat-lysed defibrinated sheep blood [New Zealand Venous Supplies, Tuakau, New Zealand]) by incubation at 37°C either aerobically, in air supplemented with 5% CO 2 , or in an anaerobic atmosphere (85% N 2 , 10% H 2 , 5% CO 2 ). For the isolation of Streptococcus pneumoniae, sheep blood agar (Columbia agar base containing 5% defibrinated sheep blood) with 5 g/ml gentamicin (Sigma, St. Louis, MO) was used with anaerobic incubation at 37°C. Representative bacterial isolates were then identified by

A comprehensive review of the nasal microbiome in chronic rhinosinusitis (CRS)

“…This study detected S.aureus and S. pneumoniae in 4 out 6 patients, the parallel cultures showed positive culture for at least one bacterium in all samples and was consistent with the PCR-DGGE results. The comparison between PCR-DGGE and cultures showed that PCR based method was able to detect a greater diversity of bacteria [46]. …”

Microbiome of the Paranasal Sinuses: Update and Literature Review

“…DNA fragments from a sample are amplified with PCR; products are then subject to gel electrophoresis and increasing concentrations of denaturing reagents; unique DNA sequences will then migrate and melt at different positions along the gel 40 the Illumina MiSeq platform (Illumina Corporation, San Diego, CA) currently generates paired sequence reads of 2 ϫ 300 bps with higher and cheaper outputs than traditional Sanger sequencing. NextSeq and HiSeq platforms (Illumina) currently enable 2 ϫ 150 bp reads.…”

“…Multiple studies have begun to characterize the microorganisms that constitute the sinus microbiome. 7,12,24,[39][40][41][42][43][44][45][46] Molecular diagnostics demonstrated that sinonasal microbial constituents differ significantly in health versus disease (Table 2). To assess the bacteriology of sinus cavities under normal conditions, Ramakrishnan et al 7 examined middle meatal swabs from 28 healthy patients.…”

Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes

“…Culture-based approaches capture <15% of resident bacterial taxa when compared to nucleic acidbased techniques, since fast-growing bacteria like staphylococci tend to predominate in culture specimens, and recovery of anaerobes and slow-growing bacteria is limited [29,33,34]. Comparing across recent surveys of the sinonasal bacterial community reveals broadly similar results, but few specific assertions can be made; agreement between studies and results have been limited by an inability to distinguish bacteria at the species level [35][36][37][38][39] but as discussed above does not give a complete reflection of the microbial community. Thus, despite the vastly superior ability of molecular techniques to identify bacterial phylotypes, species-specific identification of bacteria remains superior in culture-based techniques [40].…”