A Natural Plasmid Uniquely Encodes Two Biosynthetic Pathways Creating a Potent Anti-MRSA Antibiotic

Background Understanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin. Methodology/Principal Findings High throughput DNA sequencing of the complete genome of the producer bacterium revealed a novel 97 kb plasmid, pTML1, consisting almost entirely of two distinct gene clusters. Targeted gene knockouts confirmed the role of these clusters in biosynthesis of the two separate components, pseudomonic acid and the pyrrothine, and identified a putative amide synthetase that joins them together. Feeding mupirocin to a mutant unable to make the endogenous pseudomonic acid created a novel hybrid with the pyrrothine via “mutasynthesis” that allows inhibition of mupirocin-resistant isoleucyl-tRNA synthetase, the mupirocin target. A mutant defective in pyrrothine biosynthesis was also able to incorporate alternative amine substrates. Conclusions/Significance Plasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA

Periodontal treatment causes a longitudinal increase in nitrite-producing bacteria

BackgroundThe oral microbiome-dependent nitrate (NO3−)–nitrite (NO2−)–nitric oxide (NO) pathway may help regulate blood pressure. NO2−-producing bacteria in subgingival plaque are reduced in relative abundance in patients with untreated periodontitis compared with periodontally healthy patients. In periodontitis patients, the NO2−-producing bacteria increase several months after periodontal treatment. The early effects of periodontal treatment on NO2−-producing bacteria and the NO3−–NO2−-NO pathway remain unknown. The aim of this study was to determine how periodontal treatment affects the oral NO2−-producing microbiome and salivary NO3− and NO2− levels over time.MethodsThe subgingival microbiota of 38 periodontitis patients was analysed before (baseline [BL]) and 1, 7 and 90 days after periodontal treatment. Changes in NO2−producing bacteria and periodontitis-associated bacteria were determined by 16s rRNA Illumina sequencing. Saliva samples were collected at all-time points to determine NO3− and NO2− levels using gas-phase chemiluminescence.ResultsA significant increase was observed in the relative abundance of NO2−-producing species between BL and all subsequent timepoints (all p &lt; 0.001). Periodontitis-associated species decreased at all timepoints, relative to BL (all p &lt; 0.02). NO2−-producing species negatively correlated with periodontitis-associated species at all timepoints, with this relationship strongest 90 days post-treatment (ρ = −0.792, p &lt; 0.001). Despite these findings, no significant changes were found in salivary NO3− and NO2− over time (all p &gt; 0.05).ConclusionsPeriodontal treatment induced an immediate increase in the relative abundance of health-associated NO2−-producing bacteria. This increase persisted throughout periodontal healing. Future studies should test the effect of periodontal treatment combined with NO3− intake on periodontal and cardiovascular health.</p

Nitrate reduction capacity of the oral microbiota is impaired in periodontitis: potential implications for systemic nitric oxide availability

The reduction of nitrate to nitrite by the oral microbiota has been proposed to be important for oral health and results in nitric oxide formation that can improve cardiometabolic conditions. Studies of bacterial composition in subgingival plaque suggest that nitrate-reducing bacteria are associated with periodontal health, but the impact of periodontitis on nitrate-reducing capacity (NRC) and, therefore, nitric oxide availability has not been evaluated. The current study aimed to evaluate how periodontitis affects the NRC of the oral microbiota. First, 16S rRNA sequencing data from five different countries were analyzed, revealing that nitrate-reducing bacteria were significantly lower in subgingival plaque of periodontitis patients compared with healthy individuals (P &lt; 0.05 in all five datasets with n = 20–82 samples per dataset). Secondly, subgingival plaque, saliva, and plasma samples were obtained from 42 periodontitis patients before and after periodontal treatment. The oral NRC was determined in vitro by incubating saliva with 8 mmol/L nitrate (a concentration found in saliva after nitrate-rich vegetable intake) and compared with the NRC of 15 healthy individuals. Salivary NRC was found to be diminished in periodontal patients before treatment (P &lt; 0.05) but recovered to healthy levels 90 days post-treatment. Additionally, the subgingival levels of nitrate-reducing bacteria increased after treatment and correlated negatively with periodontitis-associated bacteria (P &lt; 0.01). No significant effect of periodontal treatment on the baseline saliva and plasma nitrate and nitrite levels was found, indicating that differences in the NRC may only be revealed after nitrate intake. Our results suggest that an impaired NRC in periodontitis could limit dietary nitrate-derived nitric oxide levels, and the effect on systemic health should be explored in future studies

The systemic inflammatory response following hand instrumentation versus ultrasonic instrumentation—a randomized controlled trial

Objective: This study sought to investigate whether the immediate systemic inflammatory response following full mouth debridement differs following use of hand compared with ultrasonic instruments. Methods: Thirty‐nine periodontitis patients were randomised to treatment with full‐mouth debridement using either hand or ultrasonic instrumentation completed within 24 hours. Serum and periodontal clinical parameters were collected at baseline, day 1, day 7 and day 90 post‐treatment. Differences in systemic inflammatory markers were assessed using general linear models at each time‐point, corrected for age, gender, smoking status, body mass index and baseline levels of each marker. Results: Across all patients, serum C‐reactive protein increased at day 1, with no differences between hand and ultrasonic groups (p(adjusted)=0.22). There was no difference between groups in interleukin‐6 (p(adjusted)=0.29) or tumour necrosis factor α (p(adjusted)=0.53) at day 1. Inflammatory markers returned to baseline levels by day 7. Treatment resulted in equal and marked improvements in clinical parameters in both groups; however, total treatment time was on average shorter for ultrasonic instruments (p(adjusted)=0.002). Conclusions: Ultrasonic instrumentation resulted in shorter treatment time with comparable clinical outcomes. Levels of serum C‐reactive protein at day 1 were similar following debridement with hand or ultrasonic instruments

Publisher Correction: Nitrate reduction capacity of the oral microbiota is impaired in periodontitis: potential implications for systemic nitric oxide availability

No abstract available

A randomized controlled trial of methotrexate for patients with generalized myasthenia gravis

OBJECTIVE: To determine the steroid-sparing effect of methotrexate (MTX) in patients with symptomatic generalized myasthenia gravis (MG). METHODS: We performed a 12-month multicenter, randomized, double-blind, placebo-controlled trial of MTX 20 mg orally every week vs placebo in 50 acetylcholine receptor antibody-positive patients with MG between April 2009 and August 2014. The primary outcome measure was the prednisone area under the dose-time curve (AUDTC) from months 4 to 12. Secondary outcome measures included 12-month changes of the Quantitative Myasthenia Gravis Score, the Myasthenia Gravis Composite Score, Manual Muscle Testing, the Myasthenia Gravis Quality of Life, and the Myasthenia Gravis Activities of Daily Living. RESULTS: Fifty-eight patients were screened and 50 enrolled. MTX did not reduce the month 4-12 prednisone AUDTC when compared to placebo (difference MTX - placebo: -488.0 mg, 95% confidence interval -2,443.4 to 1,467.3, p = 0.26); however, the average daily prednisone dose decreased in both groups. MTX did not improve secondary measures of MG compared to placebo over 12 months. Eight participants withdrew during the course of the study (1 MTX, 7 placebo). There were no serious MTX-related adverse events. The most common adverse event was nonspecific pain (19%). CONCLUSIONS: We found no steroid-sparing benefit of MTX in MG over 12 months of treatment, despite being well-tolerated. This study demonstrates the challenges of conducting clinical trials in MG, including difficulties with recruitment, participants improving on prednisone alone, and the need for a better understanding of outcome measure variability for future clinical trials. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for patients with generalized MG MTX does not significantly reduce the prednisone AUDTC over 12 months of therapy