b CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacterium Streptococcus mutans UA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of other S. mutans. The deletion of the cas genes of CRISPR1 (⌬C1S), CRISPR2 (⌬C2E), or both CRISPR1؉2 (⌬C1SC2E) or the removal of spacers 2 and 3 (⌬CR1SP13E) in S. mutans UA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation of S. mutans by the plasmids matching the spacers 2 and 3. Functional analysis of the cas deletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology in S. mutans. Compared to wild-type cells, the ⌬C1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ⌬C2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression of cas genes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we provide in vivo evidence that the type II-A CRISPR-Cas system of S. mutans may be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

Widely available and easily accessible testing for COVID-19 is a cornerstone of pandemic containment strategies. Nasopharyngeal swabs (NPS) are the currently accepted standard for sample collection but are limited by their need for collection devices and sampling by trained healthcare professionals. The aim of this study was to compare the performance of saliva to NPS in an outpatient setting. This was a prospective study conducted at three centers, which compared the performance of saliva and NPS samples collected at the time of assessment center visit. Samples were tested by real-time reverse transcription polymerase chain reaction and sensitivity and overall agreement determined between saliva and NPS. Clinical data was abstracted by chart review for select study participants. Of the 432 paired samples, 46 were positive for SARS-CoV-2, with seven discordant observed between the two sample types (four individuals testing positive only by NPS and three by saliva only). The observed agreement was 98.4% (kappa coefficient 0.91) and a composite reference standard demonstrated sensitivity of 0.91 and 0.93 for saliva and NPS samples, respectively. On average, the Ct values obtained from saliva as compared to NPS were higher by 2.76. This study demonstrates that saliva performs comparably to NPS for the detection of SARS-CoV-2. Saliva was simple to collect, did not require transport media, and could be tested with equipment readily available at most laboratories. The use of saliva as an acceptable alternative to NPS could support the use of widespread surveillance testing for SARS-CoV-2.

Background: Widespread testing for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) is necessary to curb the spread of coronavirus disease 2019 (COVID-19), but testing is undermined when the only option is a nasopharyngeal swab. Self-collected swab techniques can overcome many of the disadvantages of a nasopharyngeal swab, but they require evaluation. Methods: Three self-collected non-nasopharyngeal swab techniques (saline gargle, oral swab and combined oral-anterior nasal swab) were compared to a nasopharyngeal swab for SARS-CoV-2 detection at multiple COVID-19 assessment centers in Toronto, Canada. The performance characteristics of each test were assessed. Results: The adjusted sensitivity of the saline gargle was 0.90 (95% CI 0.86-0.94), the oral swab was 0.82 (95% CI, 0.72–0.89) and the combined oral–anterior nasal swab was 0.87 (95% CI, 0.77–0.93) compared to a nasopharyngeal swab, which demonstrated a sensitivity of ˜90% when all positive tests were the reference standard. The median cycle threshold values for the SARS-CoV-2 E-gene for concordant and discordant saline gargle specimens were 17 and 31 (P < .001), for the oral swabs these values were 17 and 28 (P < .001), and for oral–anterior nasal swabs these values were 18 and 31 (P = .007). Conclusions: Self-collected saline gargle and an oral–anterior nasal swab have a similar sensitivity to a nasopharyngeal swab for the detection of SARS-CoV-2. These alternative collection techniques are cheap and can eliminate barriers to testing, particularly in underserved populations.

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Background and objectivesPeriodontitis affects the supporting structures of the teeth as a result of the interactions between the subgingival biofilm and the host immune system. Periodontal therapy in severe forms of periodontitis often utilizes antimicrobial agents with some potential to improve host defense responses. In the present study, we investigated the in vitro effect of metronidazole (MTZ) at concentrations achievable in the periodontal pocket on PMN activation and PMN mediated killing of Porphyromonas gingivalis.Materials and methodsFlow cytometry based assays were used to measure the impact of MTZ on PMN degranulation, neutrophil extracellular trap (NET) formation and myeloperoxidase (MPO) release and phagocytosis in response to the keystone oral pathogen P. gingivalis. Functional assays for PMN mediated killing of P. gingivalis and reactive oxygen species (ROS) production in PMN were also carried out.ResultsWe demonstrate that PMNs pretreated with MTZ (2 μg/ml or 50 μg/ml) displayed enhanced killing of P. gingivalis compared to untreated PMNs. At concentrations achieved physiologically in the periodontal pocket, MTZ induced PMN surface expression of two activation markers (CD66 and CD63). MTZ did not alter P. gingivalis-induced NETosis, but suppressed P. gingivalis-induced ROS production and phagocytosis.ConclusionMTZ displays a positive interaction with PMNs to potentiate PMN mediated killing of P. gingivalis and may therefore contribute to its beneficial effects in the treatment of periodontitis initiated by P. gingivalis infections including those refractory to conventional treatment.