Investigating the Function and Regulation of Two CRISPR Systems of Streptococcus mutans

CRISPR-Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) provide adaptive microbial immunity systems against invading viruses and natural transformation via plasmids. Naturally competent Streptococcus mutans UA159 harbors two CRISPR-Cas systems: type II-A system (CRISPR1) and type I-C system (CRISPR2) and several spacers matching sequences of phage M102 or genomic sequences of other S. mutans strains. In addition, previous transcriptome studies in S. mutans linked CRISPR/Cas systems to stress response and virulence. The goal of this work was to determine the role of CRISPR/Cas systems in phage defense and natural transformation in S. mutans, and also to investigate if they play additional functions in the cell physiology. Deletion of CRISPR1 and/or CRISPR2 cas genes or removal of the spacers in S. mutans UA159 did not affect its M102 phage resistant phenotype, suggesting that CRISPR-independent mechanisms contribute to the phage resistance. Using a plasmid-based interference assay, we identified DNA interference activity in S. mutans UA159, which is mediated by its type II-A CRISPR/Cas system (CRISPR1). Spacers 2 and 3 from type II-A, both matching sequences from phage M102, were found to be essential for CRISPR interference against engineered plasmids containing matching proto-spacer sequences. Functional analysis of the cas deletion mutants revealed that CRISPR1-Cas system modulates stress tolerance induced by low pH, high temperature, oxidative and cell membrane stress, as well as DNA damaging conditions, whereas the CRISPR2-Cas participates in the tolerance associated with heat shock. Transcriptional analysis identified that VicR/K two-component signal transduction system differentially regulates the expression of cas genes for both systems in S. mutans. Further, structural, biochemical and functional studies found that the Cas5d protein SMU.1763c, a putative endoribonuclease associated with the CRISPR system I-C, acts on structured RNA substrates that is likely to be involved in CRISPR RNA processing but not in sensing cell envelope stress or preserving cell integrity in S. mutans. Together our data provide in vivo evidence that the CRISPR-Cas systems of S. mutans play novel roles in resistance against incoming plasmids that carry matching protospacer sequences and stress response.Ph.D

HPV and HIV Coinfection in Women from a Southeast Region of Romania—PICOPIV Study

Background and Objectives: Romania faces one of the highest cervical cancer burdens in Europe though it is a preventable cancer through population screening by cytology and human papillomavirus (HPV) detection. Also, it has one of the highest incidences of human immunodeficiency virus (HIV) infection. HPV and HIV coinfection are frequently encountered. The aim of study was to establish the prevalence of HPV infection among HIV-positive women in Southeast Region of Romania, to genotype high risk HPV types -and to correlate the results with clinical data and cytological cervical lesions. Materials and Methods: 40 HIV-positive women were screened for HPV types and for cytological cervical lesions. The findings were evaluated in correlation with CD4 cell counts, HIV viral load, age at first sexual intercourse, number of sexual partners, vaginal candidiasis, and Gardnerella using statistical methods. Results: 19/40 (47.5%) women were positive for HPV types, 63.15% infected with single HPV type and 36.85% with multiple HPV types. The most frequent types were type: 31 (42.1%), 56 (31.57%), 53 (15.78%). On cytology, 34 (85%) women were found with NILM of which 38.23% were HPV-positive. Fifteen percent of women had abnormal cytology (three ASC-US, three LSIL), and all of them were HPV-positive. Through analyzing the value of CD4 count, women with CD4 count ≤ 200 cells/μL were found to be significantly more likely to be infected with HPV; meanwhile there was no correlation between the detection of HPV types and HIV viral load. Candida or Gardnerella were more often associated with HIV-positive women with HPV, than in women without HPV. Conclusions: Infection with HPV types is common among HIV-positive women in the Southeast Region of Romania and it is associated with age at the beginning of sexual life, number of sexual partners, CD4 value, vaginal candidiasis, and Gardnerella infection

Detection of SARS-CoV-2 from Saliva as Compared to Nasopharyngeal Swabs in Outpatients

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