Antimicrobial and cytotoxic activity of electrosprayed chitosan nanoparticles against endodontic pathogens and Balb/c 3T3 fibroblast cells

The aims of this study were to synthesize highly positively charged chitosan nanoparticles (Ch-Np) using the electrospraying technique, and to test their antimicrobial activity against endodontic pathogens, and cytotoxicity against fibroblast cells. Ch-Np were synthesized from low molecular weight chitosan (LMW-Ch) using the electrospraying technique, and characterized. The antimicrobial activity was evaluated against Streptococcus mutans, Enterococcus faecalis, and Candida albicans in their planktonic state using a Time-Kill Test performed by using broth micro-dilution technique, and against biofilm biomass using a microtiter plate biofilm assay. The cytotoxicity was evaluated using Balb/c 3T3 fibroblast cells with the standard MTT assay. Electrospraying of LMW-Ch produced Ch-Np with an average size of 200 nm, and a surface charge of 51.7 mV. Ch-Np completely eradicated S. mutans and E. faecalis in the planktonic state and showed fungistatic activity against C. albicans. Furthermore, it significantly reduced the biofilm biomass for all the tested microbial species [S. mutans (p = 0.006), E. faecalis (p < 0.0001), and C. albicans (p = 0.004)]. When tested for cytotoxicity using 3T3 cells, Ch-Np showed no cytotoxicity. In conclusion, the highly positively charged, colloidal dispersion of Ch-Np are effective as a biocompatible endodontic antimicrobial agent.

Phage Activity Against Planktonic and Biofilm Staphylococcus aureus Periprosthetic Joint Infection Isolates

We recently reported the successful treatment of a case of periprosthetic joint infection (PJI) with phage. Phage activity against bacteria causing PJI has not been systematically evaluated. Here we examined the in vitro activity of seven lytic phages against 122 clinical isolates of Staphylococcus aureus recovered between April 1999 and February 2018 from subjects with PJI. Phages were assessed against planktonic and biofilm phenotypes. Activity of individual phages was demonstrated against up to 73% of bacterial isolates in the planktonic state and up to 100% of biofilms formed by isolates that were planktonically phage-susceptible. Susceptibility to phage was not correlated with small colony variant status. These results demonstrate that phages can infect S. aureus causing PJI in both planktonic and biofilm phenotypes, and thus are worthy of investigation as an alternative or addition to antibiotics in this setting.

Antimicrobial-resistance gene transfer of Campylobacter jejuni in mono- and dual-species biofilms

Horizontal gene transfer (HGT) is a driving force for the dissemination of antimicrobial resistance (AMR) genes among C. jejuni, a leading cause of foodborne gastroenteritis worldwide Although HGT is well documented for C. jejuni planktonic cells, the role of C. jejuni biofilms in AMR spread that likely to occur in the environment is poorly understood. Here, we developed a co-cultivation model to investigate the HGT of chromosomally-encoded AMR genes between two C. jejuni F38011 AMR mutants in biofilms. Compared to planktonic cells, C. jejuni biofilms significantly promoted HGT (P < 0.05), resulting in an increase of HGT frequencies by up to 17.5 fold. Dynamic study revealed that HGT in biofilms increased at the early stage (i.e., from 24 h to 48 h) and remained stable during 48-72 h. Biofilms continuously released the HGT mutants into supernatant culture, indicating spontaneous dissemination of AMR to broader niches. DNase I treatment confirmed the role of natural transformation in genetic exchange. HGT was not associated with biofilm biomass, cell density, or bacterial metabolic activity, whereas the presence of extracellular DNA was negatively correlated to the altered HGT frequencies. Besides, HGT in biofilms had a strain-to-strain variation. A synergistic HGT effect was observed between C. jejuni with different genomic backgrounds (i.e., C. jejuni NCTC 11168 chloramphenicol-resistant strain and F38011 kanamycin-resistant strain). C. jejuni performed HGT at the frequency of 10−7 in Escherichia coli-C. jejuni biofilms, while HGT was not detectable in Salmonella enterica-C. jejuni biofilms. IMPORTANCE Antimicrobial-resistant C. jejuni has been listed as a high priority of public health concern worldwide. To tackle the rapid evolution of AMR in C. jejuni, it is of great importance to understand the extent and characteristics of HGT in C. jejuni biofilms, which serve as the main survival strategy of this microbe in the farm-to-table continuum. In this study, we demonstrated that biofilms significantly enhanced HGT when compared to the planktonic state (P < 0.05). Biofilm cultivation time and extracellular DNA (eDNA) amount were related to varied HGT frequencies. C. jejuni could spread AMR genes in both mono-species and dual-species biofilms, mimicking the survival mode of C. jejuni in food chains. These findings indicated that the risk and extent of AMR transmission among C. jejuni have been underestimated, as previous HGT studies mainly focused on the planktonic state. Future AMR controlling measures can target biofilms and their main component eDNA.

Induction of a Viable but Nonculturable State, Thermal and Sanitizer Tolerance, and Gene Expression Correlation with the Desiccation Adapted Biofilm and Planktonic Salmonella in Powdered Infant Formula

This study investigated the effects of the physiological state, desiccation-adaptation, and storage of powdered infant formula (PIF) on Salmonella survival and their desiccation stress-related gene expression. PIF was inoculated with S . Typhimurium in the biofilm state on beads and in the planktonic state on nitrocellulose filters and stored at 25°C for up to 270 days. 5-cyano-2,3-ditoyl tetrazolium chloride flow cytometry (CTC-FCM) and Xylose Lysine Deoxycholate (XLD) plate count experiments demonstrated that biofilm-forming Salmonella tends to enter the Viable but Nonculturable (VBNC) state (p&lt; 0.05). The population reduction of all desiccation-adapted S. Typhimurium decreased significantly in both physiological states after exposure to mild heat (60℃) compared with non-adapted control cells (p&lt; 0.05). Salmonella showed heat cross-protection in both physiological states, but only the planktonic state Salmonella induced cross-protection against hydrogen peroxide. As indicated by quantitative reverse transcription polymerase chain reaction (RT-qPCR), rpoS of biofilm Salmonella for all days desiccation adatption examined and plantonic Salmonella on the 7th day of dry storage were significantly upregulated (p&lt; 0.05).The rpoE , grpE , and invA genes in S almonella of both physiological states were significantly downregulated (p&lt; 0.05). Physiological state and storage time might affect expression levels of these genes. In conclusion, prior exposure to these conditions, including low a w , and the physiological state posed an impact on Salmonella in the VBNC state and their gene expression.

Intrinsic Fluoride Tolerance Regulated by a Transcription Factor

Fluoride facilitates the remineralization of dental hard tissues and affects bacterial activities. Therefore, it is extensively used as an anti-caries agent in clinical practice and daily life. Although some studies focused on understanding Streptococcus mutans’ response to fluoride, the mechanism regulating intrinsic fluoride tolerance is not yet clear. Since the TetR family of transcription factors is associated with multidrug resistance, our aim was to evaluate whether they are related to fluoride tolerance in S. mutans. A mutant library including each S. mutans TetR gene was constructed and the transcription factor fluoride related transcriptional regulator (FrtR) was identified. The in-frame deletion of the S. mutans frtR gene resulted in decreased cell viability under fluoride in both the planktonic state and single-/dual-species biofilms. This in-frame frtR mutant was used for RNA-sequencing and the fluoride related permease gene ( frtP) was found as 1 of the downstream genes directly regulated by FrtR. The recombinant FrtR protein was purified, and conserved DNA binding motifs were determined using electrophoretic mobility shift and DNase I footprinting assays. Finally, a series of mutant and complement strains were constructed to perform the minimum inhibitory concentration (MIC) assays, which indicated that frtP upregulation led to the increase of fluoride sensitivity. Collectively, our results indicate that FrtR is an important transcription factor regulating the frtP expression in S. mutans, thus affecting the intrinsic fluoride tolerance. Therefore, this study provides novel insights into a potential target to increase the S. mutans sensitivity to fluoride for a better prevention of dental caries.

Antibacterial effect of a hyperosmotic solution containing sorbate and ethanol on Enterococcus faecalis in planktonic form and as biofilm: an in vitro study

The antibacterial effect of a hyperosmotic solution containing sorbate and ethanol on E. faecalis in planktonic state and in biofilm was evaluated. Three hyperosmotic solutions (HS-A, HS-B y HS-C) were obtained from different formulations of potassium sorbate and sodium chloride, which were tested as antimicrobials against planktonic forms of E. faecalis, in McFarland standards from 0.5 to 7, using the sedimentation technique and colony forming units (CFU) count. Afterwards an E. faecalis biofilm was produced in the palatal roots of upper first molars, by a static method in 21 days; subsequently they were prepared biomechanically by the Universal Protaper system, using the hyperosmotic solution B as an irrigant to evaluate the bacterial load reduction. One pre-instrumentation sample and one post-instrumentation sample were taken, and then were processed and cultivated to count CFU. Consecutively, roots were observed by scanning electron microscopy. The hyperosmotic solution had an important antibacterial effect when used against E. faecalis in planktonic state; solutions HS-A and HS-B were effective in eliminating E. faecalis up to 7 McFarland, while a statistical difference (p˂0.001) was observed in reducing the bacterial load in the biofilm, based on the log10 CFU count. The final solution tested seemed not to harm the dentinal structure and was capable of causing morphological changes to the bacterial cell consistent with a hyperosmotic shock. Thus, the solutions tested could be an option to be considered as irrigating agents; nonetheless further research is required regarding its biocompatibility.