Azalomycin F5a Eradicates Staphylococcus aureus Biofilm by Rapidly Penetrating and Subsequently Inducing Cell Lysis.

Antimicrobial resistance has emerged as a serious threat to public health. Bacterial biofilm, as a natural lifestyle, is a major contributor to resistance to antimicrobials. Azalomycin F5a, a natural guanidine-containing polyhydroxy macrolide, has remarkable activities against Gram-positive bacteria, including Staphylococcus aureus, a major causative agent of hospital-acquired infections. To further evaluate its potential to be developed as a new antimicrobial agent, its influence on S. aureus biofilm formation was evaluated using the crystal violet method, and then its eradication effect against mature biofilms was determined by confocal laser scanning microscopy, the drop plate method, and regrowth experiments. The results showed that azalomycin F5a could significantly inhibit S. aureus biofilm formation, and such effects were concentration dependent. In addition, it can also eradicate S. aureus mature biofilms with the minimum biofilm eradication concentration of 32.0 µg/mL. As extracellular deoxyribonucleic acid (eDNA) plays important roles in the structural integrity of bacterial biofilm, its influence on the eDNA release in S. aureus biofilm was further analyzed using gel electrophoresis. Combined with our previous works, these results indicate that azalomycin F5a could rapidly penetrate biofilm and causes damages to the cell membrane, leading to an increase in DNase release and eventually eradicating S. aureus biofilm.

Silver Diammine Fluoride Usage in General Dentistry Offices in Louisiana.

Purpose: To evaluate the current knowledge and usage of silver diammine fluoride (SDF) by general dentists in Louisiana and to identify primary barriers to the imple- mentation of SDF. Methods: A 16-item survey was emailed to 1719 Louisiana Dental Association members to identify factors influencing general dentists' usage of SDF. Results: Eighty-two surveys were completed with a response rate of 4.8 percent, with 69 identified as general dentists. Over half of the respondents were male (53.6%) and their practice experience ranged from less than one year to 48 years. The majority were solo owners (43.5%) while 7.3 percent had jobs in the corporate setting. Most agreed/strongly agreed that their knowledge of SDF was from either dental journals or online resources, while fewer stated they were taught about SDF (25%) or used SDF (8%) in dental school. The majority knew the advantages and off-label usage of SDF. However, only 40 percent recognized that SDF was officially approved for tooth hypersensitivity only. The most reported perceived barrier to SDF implementation was not learning about SDF in dental school (36%). Conclusion: There is a lack of understanding of SDF usage among Louisiana general dentists. The main reason for not incorporating SDF into their practice is the lack of training in their dental education.

Impacts of a DUF2207 Family Protein on Streptococcus mutans Stress Tolerance Responses and Biofilm Formation.

Locus SMU.243 in Streptococcus mutans was annotated as a member of the DUF2207 family proteins highly conserved in all bacteria but with unknown function. To investigate its role in S. mutans physiology, a SMU.243-deficient mutant was constructed using allelic exchange mutagenesis, and the impacts of SMU.243 deletion on bacterial growth, stress tolerance response, and biofilm formation were analyzed. Compared to the wild-type UA159, S. mutans lacking SMU.243 displayed a reduced growth rate and a reduced overnight culture density (p < 0.01) when grown at low pH and in the presence of methyl viologen. Relative to the parent strain, the deficient mutant also had a reduced survival rate following incubation in a buffer of pH 2.8 (p < 0.01) and in a buffer containing hydrogen peroxide at 58 mM after 60 min (p < 0.001) and had a reduced capacity in biofilm formation especially in the presence of sucrose (p < 0.01). To study any ensuing functional/phenotypical links between SMU.243 and uppP, which is located immediately downstream of SMU.243 and encodes an undecaprenyl pyrophosphate phosphatase involved in recycling of carrier lipid undecaprenyl phosphate, a uppP deficient mutant was generated using allelic exchange mutagenesis. Unlike the SMU.243 mutant, deletion of uppP affected cell envelope biogenesis and caused major increases in susceptibility to bacitracin. In addition, two variant morphological mutants, one forming rough colonies and the other forming mucoid, smooth colonies, also emerged following the deletion of uppP. The results suggest that the SMU.243-encoded protein of the DUF2207 family in S. mutans plays an important role in stress tolerance response and biofilm formation, but unlike the downstream uppP, does not seem to be involved in cell envelope biogenesis, although the exact roles in S. mutans' physiology awaits further investigation.

Complete Genome Sequence of Streptococcus mutans 27-3, an Active Extracellular Membrane Vesicle Producer.

Here, we report the complete genome sequence of Streptococcus mutans 27-3. Isolated from a caries-active patient, 27-3 produces significantly more extracellular membrane vesicles than the commonly used laboratory strain UA159. This study provides useful information for comparative genomic analysis and better understanding of regulation of vesiculogenesis in this bacterium.

Streptococcus mutans Lacking sufCDSUB Is Viable, but Displays Major Defects in Growth, Stress Tolerance Responses and Biofilm Formation.

Streptococcus mutans appears to possess a sole iron-sulfur (Fe-S) cluster biosynthesis system encoded by the sufCDSUB cluster. This study was designed to examine the role of sufCDSUB in S. mutans physiology. Allelic exchange mutants deficient of the whole sufCDSUB cluster and in individual genes were constructed. Compared to the wild-type, UA159, the sufCDSUB-deficient mutant, Δsuf::kan r , had a significantly reduced growth rate, especially in medium with the absence of isoleucine, leucine or glutamate/glutamine, amino acids that require Fe-S clusters for biosynthesis and when grown with medium adjusted to pH 6.0 and under oxidative and nitrosative stress conditions. Relative to UA159, Δsuf::kan r had major defects in stress tolerance responses with reduced survival rate of > 2-logs following incubation at low pH environment or after hydrogen peroxide challenge. When compared to UA159, Δsuf::kan r tended to form aggregates in broth medium and accumulated significantly less biofilm. As shown by luciferase reporter fusion assays, the expression of sufCDSUB was elevated by > 5.4-fold when the reporter strain was transferred from iron sufficient medium to iron-limiting medium. Oxidative stress induced by methyl viologen increased sufCDSUB expression by > 2-fold, and incubation in a low pH environment led to reduction of sufCDSUB expression by > 7-fold. These results suggest that lacking of SufCDSUB in S. mutans causes major defects in various cellular processes of the deficient mutant, including growth, stress tolerance responses and biofilm formation. In addition, the viability of the deficient mutant also suggests that SUF, the sole Fe-S cluster machinery identified is non-essential in S. mutans, which is not known in any other bacterium lacking the NIF and/or ISC system. However, how the bacterium compensates the Fe-S deficiency and if any novel Fe-S assembly systems exist in this bacterium await further investigation.