The effect of daily usage of Listerine Cool Mint mouthwash on the oropharyngeal microbiome: a substudy of the PReGo trial.

Introduction. ListerineÒ 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.

Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae.

BACKGROUND: The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. OBJECTIVES: To characterize the genetic pathways leading to high-level azithromycin resistance. METHODS: A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. RESULTS: Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. CONCLUSIONS: This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

Molecular typing of the actin gene of Trichomonas vaginalis isolates by PCR-restriction fragment length polymorphism.

Human trichomoniasis, caused by the protozoan Trichomonas vaginalis, is a highly prevalent sexually transmitted infection. However, little is known about the degree of strain variability of T. vaginalis. A reliable classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, pathogenesis and transmission of T. vaginalis. A PCR-restriction fragment length polymorphism typing method was designed and evaluated using T. vaginalis isolates obtained after culture of vaginal specimens collected in the Democratic Republic of Congo and in Zambia. The variation of the actin gene of T. vaginalis was determined for three ATCC reference strains and 151 T. vaginalis isolates. Eight different types were identified, on the basis of the digestion patterns of the amplified actin gene, with each of the restriction enzymes HindII, MseI and RsaI. It was determined that the ATCC reference strains 30001, 30240 and 50141 were of actin genotypes G, H and E, respectively. The actin genotype type E was more common in the Democratic Republic of Congo, whereas type G was the commonest type in Zambia. Translation of the nucleotide sequence showed up to three amino acid substitutions. We developed a reproducible, sensitive and specific typing method for T. vaginalis, and were able to distinguish at least eight T. vaginalis actin genotypes. Further studies are needed to evaluate the method using clinical specimens and to determine the utility of the typing method for the genotypic characterization of T. vaginalis.

Detection of Pentatrichomonas hominis DNA in biological specimens by PCR.

AIM: To develop a sensitive and specific polymerase chain reaction for the detection of Pentatrichomonas hominis in biological specimens. METHODS: Three primers, associated in two primer pairs, were designed to amplify a sequence from the SSU rRNA gene of P. hominis. The specificity of both primer pairs was established by testing DNA extractions of different Trichomonad species, protozoa, bacteria, yeasts, and human leucocytes. The analytical sensitivity was determined through testing dilutions of P. hominis trophozoites. The clinical specificity and applicability of the assay was evaluated on stool samples and self-administered vaginal swabs. CONCLUSIONS: A highly specific and sensitive PCR assay was developed. Both primer pairs performed equally well. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of P. hominis in vaginal specimens has not been reported before.

Comparison of culture and different PCR assays for detection of Trichomonas vaginalis in self collected vaginal swab specimens.

OBJECTIVES: DNA amplification techniques have become widely used for the diagnosis of sexually transmitted infections. For the detection of Trichomonas vaginalis, PCR techniques are not yet widely used despite the publication of several assays. The sensitivity and specificity of five independent primer sets were determined on self collected vaginal specimens obtained from female commercial sex workers. METHODS: Self collected specimens were obtained from symptomatic and asymptomatic women attending a female sex workers clinic in Abidjan, Côte d'Ivoire. Two vaginal specimens were collected, the first one was processed for culture and the second was processed for PCR analysis. PCR techniques for trichomonads were performed, using the primers as reported by Riley (TVA5/TVA6), Kengne (TVK3/TVK7), Madico (BTUB 9/BTUB 2), Shiao (IP1/IP2), and Mayta (TV1/TV2). An EIA amplicon detection method was designed for each of the primer sets. RESULTS: True positive specimens were defined as culture positive and/or two positive PCR results with EIA amplicon detection in any combination. According to this definition a prevalence of 20% was obtained compared to 7% obtained by culture. The PCR primer set TVK3/TVK7 gave the highest sensitivity (89.2%). Poor sensitivities were obtained with the primer sets TV1/TV2 (60.2%) and TVA5/TVA6 (63.9%). PCR showed a sensitivity improvement of 2.4% up to 12% when EIA was used for amplicon detection. CONCLUSIONS: Overall, the sensitivities of the different PCR assays resulting from this study were lower than those previously described. These findings could be the result of the nature of the specimen population and suggests a strain variability.

Antimicrobial susceptibilities of Neisseria gonorrhoeae in Kigali, Rwanda, and trends of resistance between 1986 and 2000.

BACKGROUND: Plasmid-mediated and chromosomal-mediated resistance of Neisseria gonorrhoeae to penicillin, tetracycline, thiamphenicol, and trimethoprim-sulfamethoxazole has spread dramatically in Africa. Monitoring of antimicrobial susceptibility is a key element in the control of sexually transmitted diseases. GOAL: To document antimicrobial susceptibilities of gonococci isolated during the past 15 years in Kigali, Rwanda. STUDY DESIGN: Minimal inhibitory concentrations of recently collected gonococcal isolates of eight antimicrobials were determined. The results were compared with data collected for isolates obtained since 1986. RESULTS: In 1986, 35% of the gonococcal isolates were penicillinase-producing N gonorrhoeae. Tetracycline-resistant N gonorrhoeae appeared in 1989. The prevalence of penicillinase-producing N gonorrhoeae and tetracycline-resistant N gonorrhoeae increased significantly to 70.5% and 89.2%, respectively. Chromosomal resistance to penicillin, tetracycline, and thiamphenicol increased temporarily, then decreased significantly. Chromosomal resistance to trimethoprim-sulfamethoxazole appeared in 1988 and increased to 21.6%. All the isolates were susceptible to ceftriaxone, ciprofloxacin, spectinomycin, and kanamycin. CONCLUSIONS: This study illustrated the rapidly increasing frequencies of penicillinase-producing N gonorrhoeae and tetracycline-resistant N gonorrhoeae. Chromosomal resistance to thiamphenicol and trimethoprim-sulfamethoxazole excludes these drugs as alternative treatment. Programs for antimicrobial susceptibility surveillance of N gonorrhoeae should urgently be established in Africa.

Antimicrobial susceptibilities and plasmid patterns of Neisseria gonorrhoeae in Bénin.

This study describes antimicrobial susceptibility patterns of Neisseria gonorrhoeae isolates obtained from female sex workers in Cotonou, Bénin. All isolates were susceptible to spectinomycin, ceftriaxone and ciprofloxacin, and susceptible to moderately susceptible to kanamycin; 9.8% of isolates were resistant to thiamphenicol; 9%, 87.5% and 3.5% were susceptible, moderately susceptible, resistant to trimethoprim-sulfamethoxazole, respectively; 94.4% and 99.3% were resistant to penicillin and tetracycline, respectively. All isolates with a minimal inhibitory concentration of tetracycline of >8 mg/l carried the 'American type' tetM plasmid; 94% and 6% of penicillinase-producing isolates possessed a 3.2 MDa and a 4.4MDa beta-lactamase plasmid, respectively. Surveillance of antimicrobial susceptibility of N. gonorrhoeae isolates to currently used drugs in Africa should become part of sexually transmitted diseases (STDs) control programmes.

Development of a heminested polymerase chain reaction assay for the detection of Haemophilus ducreyi in clinical specimens.

Detection of Haemophilus ducreyi in genital ulcer specimens by culture lacks sensitivity. To enhance detection, a heminested polymerase chain reaction (PCR) assay was developed targeting the nucleotide sequence of a gene, designated p27, which encodes for a 27 kDa H. ducreyi-specific protein. The p27 PCR assay detected all (37/37) H. ducreyi strains tested and gave no amplified product from DNA extracts of any of 31 other microorganisms, from 30 non-genital ulcer specimens, or from 29 urethral and vaginal swab specimens collected from non-chancroid STD patients. In genital ulcer disease specimens, compared to combined positive results obtained by culture and a previously described PCR assay, the p27 PCR assay showed a sensitivity of 91% (48/53). The p27 PCR assay provides a specific and a sensitive detection of H. ducreyi in clinical specimens.