- Volume 73, Issue 6, 2024

Introduction. Listerine is a bactericidal mouthwash widely used to prevent oral health problems such as dental plaque and gingivitis. However, whether it promotes or undermines a healthy oral microbiome is unclear.

Hypothesis/Gap Statement. We hypothesized that the daily use of Listerine Cool Mint would have a significant impact on the oropharyngeal microbiome.

Aim. We aimed to assess if daily usage of Listerine Cool Mint influenced the composition of the pharyngeal microbiome.

Methodology. The current microbiome substudy is part of the Preventing Resistance in Gonorrhoea trial. This was a double-blind single-centre, crossover, randomized controlled trial of antibacterial versus placebo mouthwash to reduce the incidence of gonorrhoea/chlamydia/syphilis in men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP). Fifty-nine MSM taking HIV PrEP were enrolled. In this crossover trial, participants received 3 months of daily Listerine followed by 3 months of placebo mouthwash or vice versa. Oropharyngeal swabs were taken at baseline and after 3 months use of each mouthwash. DNA was extracted for shotgun metagenomic sequencing (Illumina Inc.). Non-host reads were taxonomically classified with MiniKraken and Bracken. The alpha and beta diversity indices were compared between baseline and after each mouthwash use. Differentially abundant bacterial taxa were identified using ANOVA-like differential expression analysis.

Results. Streptococcus was the most abundant genus in most samples (n = 103, 61.7 %) with a median relative abundance of 31.5% (IQR 20.6–44.8), followed by Prevotella [13.5% (IQR 4.8–22.6)] and Veillonella [10.0% (IQR 4.0–16.8)]. Compared to baseline, the composition of the oral microbiome at the genus level (beta diversity) was significantly different after 3 months of Listerine (P = 0.006, pseudo-F = 2.29) or placebo (P = 0.003, pseudo-F = 2.49, permutational multivariate analysis of variance) use. Fusobacterium nucleatum and Streptococcus anginosus were significantly more abundant after Listerine use compared to baseline.

Conclusion. Listerine use was associated with an increased abundance of common oral opportunistic bacteria previously reported to be enriched in periodontal diseases, oesophageal and colorectal cancer, and systemic diseases. These findings suggest that the regular use of Listerine mouthwash should be carefully considered.

Introduction. Colorectal cancer (CRC) is a leading cause of cancer deaths, closely linked to the intestinal microbiota and bile acid metabolism. Secondary bile acids, like deoxycholic and lithocholic acid, are associated with increased CRC risk due to their disruption of vital cellular functions. In contrast, isoallolithocholic acid (isoalloLCA) shows potential health benefits, highlighting the complex role of bile acids in CRC. A specific primer set was previously developed to amplify homologs of the 5α-reductase gene (5ar), which are involved in the biosynthesis of isoalloLCA, thereby enabling the estimation of abundance of 5ar (5ar levels) in the intestine.

Hypothesis/Gap Statement. We hypothesized that 5ar levels in the intestine are associated with CRC.

Aim. This study aimed to investigate intestinal 5ar levels and compare them across different stages of the adenoma–carcinoma sequence, providing insights into novel strategies for monitoring CRC risk.

Methodology. DNA was extracted from intestinal lavage fluids (ILF) collected during 144 colonoscopies. Next-generation sequencing (NGS) was employed to examine the sequence of 5ar homologues, using a specific primer set on DNA from seven selected ILFs – four from carcinoma patients and three from individuals with non-neoplastic mucosa. Additionally, we used quantitative PCR (qPCR) to measure 5ar levels in all 144 DNA samples.

Results. We conducted 144 colonoscopies and categorized patients according to the adenoma–cancer sequence: 52 with non-neoplastic mucosa, 69 with adenomas and 23 with carcinoma. Analysis of 292,042 NGS-derived 5ar sequences revealed the seven most prevalent amplicon sequence variants, each 254 base pairs in length. These closely matched or were identical to 5ar sequences in Bacteroides uniformis, Phocaeicola vulgatus and Phocaeicola dorei. Furthermore, qPCR analysis demonstrated significantly lower 5ar levels in the carcinoma group compared to those in the non-neoplastic mucosa group (P = 0.0004). A similar, though not statistically significant, trend was observed in the adenoma group (P = 0.0763), suggesting that 5ar levels decrease as CRC progresses.

Conclusion. These findings indicate that PCR-based monitoring of 5ar levels in intestinal samples over time could provide a non-invasive, rapid and cost-effective method for assessing an increased risk of CRC.

Introduction. Avian reovirus (ARV) is associated with arthritis/tenosynovitis and malabsorption syndrome in chickens. The σC and σB proteins, both exposed to the virus capsid, are highly immunogenic and could form the basis for diagnostic devices designed to assess the immunological status of the flock.

Gap Statement. Commercial ARV ELISAs cannot distinguish between vaccinated and infected animals and might not detect circulating ARV strains.

Aim. We aimed to develop a customized test to detect the circulating field ARV strains as well as distinguish between vaccinated and unvaccinated animals.

Methodology. We developed ELISA assays based on recombinant (r) σB, σC and the nonstructural protein σNS and tested them using antisera of vaccinated and unvaccinated chickens as well as negative controls. Fragments of σB and σC proteins were also used to study regions that could be further exploited in diagnostic tests.

Results. Vaccinated and unvaccinated birds were positive by commercial ELISA, with no difference in optical density values. In contrast, samples of unvaccinated animals showed lower absorbance in the rσB and rσC ELISA tests and higher absorbance in the rσNS ELISA test than the vaccinated animals. Negative control samples were negative in all tests. Fragmentation of σB and σC proteins showed that some regions can differentiate between vaccinated and unvaccinated animals. For example, σB amino acids 128–179 (σB-F4) and σC amino acids 121–165 (σC-F4) exhibited 85 and 95% positivity among samples of vaccinated animals but only 5% and zero positivity among samples of unvaccinated animals, respectively.

Conclusion. These data suggest that unvaccinated birds might have been exposed to field strains of ARV. The reduction in absorbance in the recombinant tests possibly reflects an increased specificity of our test since unvaccinated samples showed less cross-reactivity with the vaccine proteins immobilized on ELISAs. The discrepant results obtained with the protein fragment tests between vaccinated and unvaccinated animals are discussed in light of the diversity between ARV strains.

Introduction. The fungal pathogen Aspergillus fumigatus can induce prolonged colonization of the lungs of susceptible patients, resulting in conditions such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis.

Hypothesis. Analysis of the A. fumigatus secretome released during sub-lethal infection of G. mellonella larvae may give an insight into products released during prolonged human colonisation.

Methodology. Galleria mellonella larvae were infected with A. fumigatus, and the metabolism of host carbohydrate and proteins and production of fungal virulence factors were analysed. Label-free qualitative proteomic analysis was performed to identify fungal proteins in larvae at 96 hours post-infection and also to identify changes in the Galleria proteome as a result of infection.

Results. Infected larvae demonstrated increasing concentrations of gliotoxin and siderophore and displayed reduced amounts of haemolymph carbohydrate and protein. Fungal proteins (399) were detected by qualitative proteomic analysis in cell-free haemolymph at 96 hours and could be categorized into seven groups, including virulence (n = 25), stress response (n = 34), DNA repair and replication (n = 39), translation (n = 22), metabolism (n = 42), released intracellular (n = 28) and cellular development and cell cycle (n = 53). Analysis of the Gallerial proteome at 96 hours post-infection revealed changes in the abundance of proteins associated with immune function, metabolism, cellular structure, insect development, transcription/translation and detoxification.

Conclusion. Characterizing the impact of the fungal secretome on the host may provide an insight into how A. fumigatus damages tissue and suppresses the immune response during long-term pulmonary colonization.