Encapsulation of Lactiplantibacillus plantarum CRD7 in sub-micron pullulan fibres by spray drying: Maximizing viability with prebiotic and thermal protectants.

Pullulan was used as the wall material for microencapsulation of L. plantarum CRD7 by spray drying, while isomalto-oligosaccharides (IMO) was used as prebiotic. Also, the effect of different thermal protectants on survival rate during microencapsulation was evaluated. Taguchi orthogonal array design showed that pullulan at 14 % concentration, IMO at 30 % concentration and whey protein isolate at 20 % rate were the optimized wall material, prebiotic and thermal protectant, respectively for microencapsulation of L. plantarum. FESEM images revealed that the spray-dried encapsulates were fibrous similar to those produce by electrospinning, while fluorescence microscopy ascertained that most of the probiotic cells were alive and intact after microencapsulation. The adsorption-desorption isotherm was of Type II and the encapsulate had specific surface area of 1.92 m2/g and mean pore diameter of 15.12 nm. The typical amide II and III bands of the bacterial proteins were absent in the FTIR spectra, suggestive of adequate encapsulation. DSC thermogram showed shifting of melting peaks to wider temperature range due to interactions between the probiotic and wall materials. IMO at 30 % (w/w) along with WPI at 20 % concentration provided the highest storage stability and the lowest rate of cell death of L. plantarum after microencapsulation. Acid and bile salt tolerance results confirmed that microencapsulated L. plantarum could sustain the harsh GI conditions with >7.5 log CFU/g viability. After microencapsulation, L. plantarum also possessed the ability to ferment milk into curd with pH of 4.62.

Impact of Prebiotic on Viability of Lactiplantibacillus plantarum D-2 by Encapsulation through Spray Drying and Its Commercialization Potential.

This study investigated the impact of inulin (INL) on viability of L. plantarum D-2 (LPD2) by encapsulation through spray drying (SD) and its commercialization potential to alternative of conventional wall material maltodextrin (MD). LPD2, derived from sea tangle (Saccharina japonica) kimchi, is probiotics exhibiting significant attributes like cholesterol reduction, antioxidant properties, and resilience to acidic and bile environments. To enhance storage viability and stability of LPD2, encapsulation was applied by SD technology. The optimum encapsulation condition with MD was 10% MD concentration (MD10) and inlet temperature (96°C). The optimum concentration ratio of MD and INL was 7:3 (INL3) for alternative of MD with similar encapsulation yield and viability of LPD2. Viability of LPD2 with INL3 exhibited almost 8% higher than that with MD10 after 50 days storage at 25°C. Physicochemical characteristics of the encapsulated LPD2 (ELPD2) with MD10 and INL3 had no significant different between flowability and morphology. But, ELPD2 with INL3 had lower water solubility and higher water absorption resulting in extension of viability of LPD2 compared to that with MD10. The comprehensive study results showed that there was no significant difference in the encapsulation yield and physicochemical properties between ELPD2 with MD10 and INL3, except of water solubility index (WSI) and water absorption index (WAI). INL have the potential to substitute of MD as a commercial wall material with prebiotic functionality to enhance the viability of LPD2 by encapsulation.

Effectiveness of experimental dentifrices based on essential oils on biofilm on complete dentures: an in vitro study.

Specific products containing natural resources can contribute to the innovation of complete denture hygiene. OBJECTIVE: To conduct an in vitro evaluation of experimental dentifrices containing essential oils of Bowdichia virgilioides Kunth (BvK), Copaifera officinalis (Co), Eucalyptus citriodora (Ec), Melaleuca alternifolia (Ma) and Pinus strobus (Ps) at 1%. METHODOLOGY: The variables evaluated were organoleptic and physicochemical characteristics, abrasiveness (mechanical brushing machine) simulating 2.5 years, and microbial load (Colony Forming Units - CFU/mL), metabolic activity (XTT assay) and cell viability (Live/Dead® BacLight™ kit) of the multispecies biofilm (Streptococcus mutans: Sm, Staphylococcus aureus: Sa, Candida albicans: Ca and Candida glabrata: Cg). Specimens of heat-polymerized acrylic resins (n=256) (n=96 specimens for abrasiveness, n=72 for microbial load count, n=72 for biofilm metabolic activity, n=16 for cell viability and total biofilm quantification) with formed biofilm were divided into eight groups for manual brushing (20 seconds) with a dental brush and distilled water (NC: negative control), Trihydral (PC: positive control), placebo (Pl), BvK, Co, Ec, Ma or Ps. After brushing, the specimens were washed with PBS and immersed in Letheen Broth medium, and the suspension was sown in solid specific medium. The organoleptic characteristics were presented by descriptive analysis. The values of density, pH, consistency and viscosity were presented in a table. The data were analyzed with the Wald test in a generalized linear model, followed by the Kruskal-Wallis test, Dunn's test (mass change) and the Bonferroni test (UFC and XTT). The Wald test in Generalized Estimating Equations and the Bonferroni test were used to analyze cell viability. RESULTS: All dentifrices showed stable organoleptic characteristics and adequate physicochemical properties. CN, Ec, Ps, Pl and PC showed low abrasiveness. There was a significant difference between the groups (p<0.001) for microbial load, metabolic activity and biofilm viability. CONCLUSIONS: It was concluded that the BvK, Ec and Ps dentifrices are useful for cleaning complete dentures, as they have antimicrobial activity against biofilm. The dentifrices containing Bowdichia virgilioides Kunth showed medium abrasiveness and should be used with caution.

Arginine and sodium fluoride affect the microbial composition and reduce biofilm metabolism and enamel mineral loss in an oral microcosm model.

OBJECTIVE: To assess the effects of arginine, with or without sodium fluoride (NaF; 1,450 ppm), on saliva-derived microcosm biofilms and enamel demineralization. METHODS: Saliva-derived biofilms were grown on bovine enamel blocks in 0.2 % sucrose-containing modified McBain medium, according to six experimental groups: control (McBain 0.2 %); 2.5 % arginine; 8 % arginine; NaF; 2.5 % arginine with NaF; and 8 % arginine with NaF. After 5 days of growth, biofilm viability was assessed by colony-forming units counting, laser scanning confocal microscopy was used to determine biofilm vitality and extracellular polysaccharide (EPS) production, while biofilm metabolism was evaluated using the resazurin assay and lactic acid quantification. Demineralization was evaluated by measuring pH in the culture medium and calcium release. Data were analyzed by Kruskal-Wallis' and Dunn's tests (p < 0.05). RESULTS: 8 % arginine with NaF showed the strongest reduction in total streptococci and total microorganism counts, with no significant difference compared to arginine without NaF. Neither 2.5 % arginine alone nor NaF alone significantly reduced microbial counts compared to the control, although in combination, a reduction in all microbial groups was observed. Similar trends were found for biofilm vitality and EPS, and calcium released to the growth medium. CONCLUSIONS: 8 % Arginine, with or without NaF, exhibited the strongest antimicrobial activity and reduced enamel calcium loss. Also, NaF enhanced the effects of 2.5 % arginine, yielding similar results to 8 % arginine for most parameters analyzed. CLINICAL SIGNIFICANCE: The results provided further evidence on how arginine, with or without NaF, affects oral microcosm biofilms and enamel mineral loss.

Deep eutectic supramolecular polymer functionalized MXene for enhancing mechanical properties, photothermal conversion, and bacterial inactivation of cellulose textiles.

Two-dimensional (2D) transition metal carbides (Ti3C2Tx MXene) have gained significant attention for their potential in constructing diverse functional materials, However, MXene is easily oxidized and weakly bound to the cellulose matrix, which pose challenges in developing MXene-decorated non-woven fabric with strong bonding and stable thermal management properties. Herein, we successfully prepared deep eutectic supramolecular polymer (DESP) functionalized MXene to address these issues. MXene can be wrapped with DESP to be insulated from water and protected from being oxidized. Subsequently, we achieved an efficient in-situ deposition of DESP-functionalized MXene onto fibers through a combination of dip coating and photopolymerization technique. The resulting nonwoven fabric (CNs-DESP@M) exhibited excellent photothermal conversion properties along with rapid thermal response and functional stability. Interestingly, the interface bonding between MXene and the fiber surface was significantly enhanced due to the abundant pyrogallol groups in DESP, resulting in the composite textile exhibiting commendable mechanical properties (2.68 MPa). Moreover, the as-prepared textile demonstrates outstanding bactericidal efficacy against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The multifunctional textile, created through a facile and efficient approach, demonstrates remarkable potential for applications in smart textiles, catering to the diverse needs of individuals in the future.

Elimination of E. faecalis with NaOCl versus chlorhexidine gluconate from primary molar root canal systems: an ex vivo model study.

OBJECTIVES: This ex vivo human study aimed to evaluate the efficacy of NaOCl and chlorhexidine gluconate (CHG) irrigations in eliminating Enterococcus faecalis from the RCS of primary molars. MATERIALS AND METHODS: Disinfected extracted primary molars were inoculated with E. faecalis for 24 h. Then, the RCS samples were then irrigated with either 2.5% NaOCl, 0.2% and 2% CHG, or sham saline. The samples were collected immediately after irrigation; and 24 h later, the bacterial viability and counts were measured using blood agar and qRT-PCR, respectively. Histological sections were used to measure E. faecalis penetration and viability in dentin tubules using fluorescence microscopy. RESULTS: The recovery of viable E. faecalis after the irrigation of the primary molars showed more significant bactericidal effects of NaOCl and 0.2% and 2% CHG than of saline. Immediately after the irrigation, the NaOCl group showed the greatest reduction in E. faecalis; and 24 h later, all the groups had lower viable E. faecalis than the saline control. The bacterial penetration was also lowest in the NaOCl group, although there was no difference in bacterial viability in the tubules between the groups. CONCLUSION: In primary teeth, NaOCl and CHG showed similar degrees of bacterial elimination efficacy in terms of E.faecalis. CLINICAL RELEVANCE: Within the limitations of this study, NaOCl and CHG have the similar ability to perform endodontic irrigation of primary ex vivo teeth regarding the elimination of E.faecalis, but NaOCl penetrates dentin tubules better.

Double-layer microcapsules based on shellac for enhancing probiotic survival during freeze drying, storage, and simulated gastrointestinal digestion.

Probiotics are susceptible to diverse conditions during processing, storage, and digestion. Here, shellac (SC), sodium alginate (SA), coconut oil (CO), soybean oil (SO), and trehalose (AL) were used to prepare microcapsules aiming to improve the survival of Lactiplantibacillus plantarum KLDS1.0318 during freeze-drying, storage process, and gastrointestinal digestion. The results showed that for SA/AL/SC/CO and SA/AL/SC/SO, the survival loss decreased by 51.2 % and 51.0 % after a freeze-drying process compared with microcapsules embedded by SA; the viable bacteria count loss decreased by 4.36 and 4.24 log CFU/mL compared with free cell (CON) during storage for 28 d under 33%RH at 25 °C, respectively; while for simulating digestion in vitro, the survival loss decreased by 3.05 and 2.70 log CFU/mL, 0.63 and 0.55 log CFU/mL after digestion at simulated gastric fluid for 120 min and small intestine fluid for 180 min, respectively (P < 0.05). After microcapsules were added to fermented dairy stored at 4 °C for 21 d, the viable bacteria count of SA/AL/SC/CO and SA/AL/SC/SO significantly increased by 2.10 and 1.70 log CFU/mL compared with CON, respectively (P < 0.05). In conclusion, the current study indicated that shellac-based probiotic microcapsules have superior potential to protect and deliver probiotics in food systems.

Construction of Fu brick tea polysaccharide-cold plasma modified alginate microgels for probiotic delivery: Enhancing viability and colonization.

Emerging food processing technologies provide broader avenues for enhancing probiotic delivery systems. In this study, the new Fu brick tea polysaccharide (FBTP) was extracted and combined with cold plasma-modified alginate nano-montmorillonite (AMT) to prepare microgels by ionic gelation to improve the viability of encapsulated Lactobacillus kefiranofaciens JKSP109. Results showed that cold plasma treatment for 3 min changed the surface charge of AMT biopolymer solution, and FBTP addition reduced the particle size to the lowest of 223 ± 5.50 nm. Morphological analysis showed that the AMT treated with cold plasma for 3 min and FBTP (C3AMT + FBTP) formed a dense microgel through electrostatic interaction, and the probiotics were randomly distributed in their internal polysaccharide network, as well as the interlayer and surrounding of nanoparticles. The probiotics immobilized in C3AMT + FBTP microgel exhibited the highest viability (8.48 ± 0.03 log CFU/g) and colonic colonization after exposure to simulated gastrointestinal conditions. In addition, the good antioxidant activity of FBTP reduced the loss of probiotic viability during storage, with only 2.58 log CFU/g decreased after 4 weeks. Therefore, such probiotic products enriched with natural bioactive ingredients can be developed as a potential functional food additive.

Ascorbic acid potentiates photodynamic inactivation mediated by octyl gallate and blue light for rapid eradication of planktonic bacteria and biofilms.

This study reported for the first time that Ascorbic acid (AA) could appreciably boost the efficiency of Octyl gallate (OG)-mediated photodynamic inactivation (PDI) on Escherichia coli and Staphylococcus aureus in planktonic and biofilm states. The combination of OG (0.075 mM) and AA (200 mM) with 420 nm blue light (212 mW/cm2) led to a >6 Log killing within only 5 min for E. coli and S. aureus and rapid eradication of biofilms. The mechanism of action appears to be the generation of highly toxic hydroxyl radicals (•OH) via photochemical pathways. OG was exposed to BL irradiation to generate various reactive oxygen radicals (ROS) and the addition of AA could transform singlet oxygen (1O2) into hydrogen peroxide (H2O2), which could further react with AA to generate enormous •OH. These ROS jeopardized bacteria and biofilms by nonspecifically attacking various biomacromolecules. Overall, this PDI strategy provides a powerful microbiological decontamination modality to guarantee safe food products.

Efficacy of 233 nm LED far UV-C-radiation against clinically relevant bacterial strains in the phase 2/ step 2 in vitro test on basis of EN 14561 and on an epidermis cell model.

INTRODUCTION: Healthcare-acquired infections and overuse of antibiotics are a common problem. Rising emergence of antibiotic and antiseptic resistances requires new methods of microbial decontamination or decolonization as the use of far-UV-C radiation. METHODS: The microbicidal efficacy of UV-C radiation (222 nm, 233 nm, 254 nm) was determined in a quantitative carrier test and on 3D-epidermis models against Staphylococcus (S.) aureus, S.epidermidis, S.haemolyticus, S.lugdunensis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. To mimic realistic conditions, sodium chloride solution, mucin, albumin, artificial saliva, artificial wound exudate and artificial sweat were used. RESULTS: In sodium chloride solution, irradiation with a dose of 40 mJ/cm2 (233 nm) was sufficient to achieve 5 lg reduction independent of bacteria genus or species. In artificial sweat, albumin and artificial wound exudate, a reduction >3 lg was reached for most of the bacteria. Mucin and artificial saliva decreased the reduction to <2 lg. On 3D epidermis models, reduction was lower than in the carrier test. CONCLUSION: UV-C radiation at 233 nm was proven to be efficient in bacteria inactivation independent of genus or species thus being a promising candidate for clinical use in the presence of humans and on skin/mucosa.

In vitro activity of apramycin (EBL-1003) in combination with colistin, meropenem, minocycline or sulbactam against XDR/PDR Acinetobacter baumannii isolates from Greece.

OBJECTIVES: To evaluate the in vitro activity of the combination of apramycin with colistin, meropenem, minocycline or sulbactam, against some well-characterized XDR Acinetobacter baumannii clinical isolates from Greece, to understand how apramycin can be best incorporated into clinical practice and optimize effectiveness. METHODS: In vitro interactions of apramycin (0.5×, 1× and 2× the MIC value) with colistin (2 mg/L), meropenem (30 mg/L), minocycline (3.5 mg/L) or sulbactam (24 mg/L) were tested using time-kill methodology. Twenty-one clinical A. baumannii isolates were chosen, exhibiting apramycin MICs of 4-16 mg/L, which were at or below the apramycin preliminary epidemiological cut-off value of 16 mg/L. These isolates were selected for a range of colistin (4-32 mg/L), meropenem (16-256 mg/L), minocycline (8-32 mg/L) and sulbactam (8-32 mg/L) MICs across the resistant range. Synergy was defined as a ≥2 log10 cfu/mL reduction compared with the most active agent. RESULTS: The combination of apramycin with colistin, meropenem, minocycline or sulbactam was synergistic, at least at one of the concentrations of apramycin (0.5×, 1× or 2× MIC), against 83.3%, 90.5%, 90.9% or 92.3% of the tested isolates, respectively. Apramycin alone was bactericidal at 24 h against 9.5% and 33.3% of the tested isolates at concentrations equal to 1× and 2× MIC, while the combination of apramycin at 2× MIC with colistin, meropenem or sulbactam was bactericidal against all isolates tested (100%). The apramycin 2× MIC/minocycline combination had bactericidal activity against 90.9% of the tested isolates. CONCLUSIONS: Apramycin combinations may have potential as a treatment option for XDR/pandrug-resistant (PDR) A. baumannii infections and warrant validation in the clinical setting, when this new aminoglycoside is available for clinical use.

Effects of Sodium Hypochlorite Concentration and Application Time on Bacteria in an Ex Vivo Polymicrobial Biofilm Model.

INTRODUCTION: In endodontic treatment, it is important to remove or inactivate biofilms in the root canal system. We investigated the effects of different concentrations and application times of sodium hypochlorite (NaOCl) on the viability of bacteria in ex vivo polymicrobial biofilms of different maturation levels. METHODS: Polymicrobial biofilms were prepared from dental plaque samples and grown for 1, 2, and 3 weeks under anaerobic conditions on collagen-coated hydroxyapatite discs as an ex vivo biofilm model. The biofilms were then exposed to NaOCl at concentrations ranging from 0.1% to 2% for 1 or 3 minutes. The control group was exposed to sterile distilled water. Viability staining was performed and examined by confocal laser scanning microscopy to determine the percentage of biofilm bacteria killed by NaOCl. Scanning electron microscopy was also performed to visually examine the biofilms. RESULTS: Application of NaOCl at 0.5%-2% for both 1 and 3 min killed significantly more bacteria when compared to the controls (P < .05). Cell viability tended to be lower after the application of NaOCl for 3 minutes than that for 1 minute. CONCLUSIONS: Our experiments using an ex vivo model showed that within the range of 0.1%-2% of NaOCl, higher NaOCl concentrations and longer application times were more effective in killing biofilm bacteria, and that mature biofilms were more resistant to NaOCl than younger biofilms.

The effect of enzymatic and viability dye treatment in combination with long-range PCR on assessing Tulane virus infectivity.

Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID50). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID50 reductions of 0.3, 4.4 and 5.9 log10 were observed. UV exposure (40/100/1000 mJ/cm2) resulted in 1.1, 2.5 and 5.9 log10 reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log10. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID50. On average, RT-qPCR with pre-treatments deviated by 1.1-1.3 log10 from TCID50. For UV light, long-range PCR was closest to TCID50 results. Long-range reductions deviated from TCID50 by ≤0.1 log10 for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log10 from TCID50. After chlorination the molecular methods repeatedly deviated from TCID50 by >1.0 log10, Overall, each method needs to be further optimized for the individual types of inactivation treatment.

Lactobacillus Persisters Formation and Resuscitation.

Lactobacillus is a commonly used probiotic, and many researchers have focused on its stress response to improve its functionality and survival. However, studies on persister cells, dormant cells that aid bacteria in surviving general stress, have focused on pathogenic bacteria that cause infection, not Lactobacillus. Thus, understanding Lactobacillus persister cells will provide essential clues for understanding how Lactobacillus survives and maintains its function under various environmental conditions. We treated Lactobacillus strains with various antibiotics to determine the conditions required for persister formation using kill curves and transmission electron microscopy. In addition, we observed the resuscitation patterns of persister cells using single-cell analysis. Our results show that Lactobacillus creates a small population of persister cells (0.0001-1% of the bacterial population) in response to beta-lactam antibiotics such as ampicillin and amoxicillin. Moreover, only around 0.5-1% of persister cells are heterogeneously resuscitated by adding fresh media; the characteristics are typical of persister cells. This study provides a method for forming and verifying the persistence of Lactobacillus and demonstrates that antibiotic-induced Lactobacillus persister cells show characteristics of dormancy, sensitivity of antibiotics, same as exponential cells, multi-drug tolerance, and resuscitation, which are characteristics of general persister cells. This study suggests that the mechanisms of formation and resuscitation may vary depending on the characteristics, such as the membrane structure of the bacterial species.

RecN spatially and temporally controls RecA-mediated repair of DNA double-strand breaks.

RecN, a bacterial structural maintenance of chromosomes-like protein, plays an important role in maintaining genomic integrity by facilitating the repair of DNA double-strand breaks (DSBs). However, how RecN-dependent chromosome dynamics are integrated with DSB repair remains unclear. Here, we investigated the dynamics of RecN in response to DNA damage by inducing RecN from the PBAD promoter at different time points. We found that mitomycin C (MMC)-treated ΔrecN cells exhibited nucleoid fragmentation and reduced cell survival; however, when RecN was induced with arabinose in MMC-exposed ΔrecN cells, it increased a level of cell viability to similar extent as WT cells. Furthermore, in MMC-treated ΔrecN cells, arabinose-induced RecN colocalized with RecA in nucleoid gaps between fragmented nucleoids and restored normal nucleoid structures. These results suggest that the aberrant nucleoid structures observed in MMC-treated ΔrecN cells do not represent catastrophic chromosome disruption but rather an interruption of the RecA-mediated process. Thus, RecN can resume DSB repair by stimulating RecA-mediated homologous recombination, even when chromosome integrity is compromised. Our data demonstrate that RecA-mediated presynapsis and synapsis are spatiotemporally separable, wherein RecN is involved in facilitating both processes presumably by orchestrating the dynamics of both RecA and chromosomes, highlighting the essential role of RecN in the repair of DSBs.

Natural cordiaquinones as strategies to inhibit the growth and biofilm formation of methicillin-sensitive and methicillin-resistant Staphylococcus spp.

AIMS: The aim of this study was to investigate the antibacterial and antibiofilm potential of cordiaquinones B, E, L, N, and O against different Staphylococci strains, in addition to analyzing in silico the observed effect. METHODS AND RESULTS: The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined according to CLSI guidelines. The inhibition of biofilm formation was investigated at sub-MICs. Atomic force microscopy (AFM) and density functional theory method were performed. The tested strains of Staphylococcus spp. were susceptible to cordiaquinones B, E, and L, among which cordiaquinone B exerted a bactericidal effect, confirmed by a bacterial growth curve study, against Staphylococcus saprophyticus. Cordiaquinones B and E showed lowest MBC values against S. saprophyticus. AFM revealed that cordiaquinone L reduced the mean cell size of S. saprophyticus. Cordiaquinones B and E inhibited the biofilm formation ability of S. aureus by ∼90%. The in silico analysis suggested that the antimicrobial activity of cordiaquinones is driven by their electron donation capability. CONCLUSIONS: Cordiaquinones inhibit the growth and biofilm formation (virulence factor) of both methicillin-sensitive and methicillin-resistant Staphylococci strains, indicating their antimicrobial potential.

Teixobactin kills bacteria by a two-pronged attack on the cell envelope.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.

Aspergillus nidulans Septa Are Indispensable for Surviving Cell Wall Stress.

Septation in filamentous fungi is a normal part of development, which involves the formation of cross-hyphal bulkheads, typically containing pores, allowing cytoplasmic streaming between compartments. Based on previous findings regarding septa and cell wall stress, we hypothesized that septa are critical for survival during cell wall stress. To test this hypothesis, we used known Aspergillus nidulans septation-deficient mutants (ΔsepH, Δbud3, Δbud4, and Δrho4) and six antifungal compounds. Three of these compounds (micafungin, Congo red, and calcofluor white) are known cell wall stressors which activate the cell wall integrity signaling pathway (CWIS), while the three others (cycloheximide, miconazole, and 2,3-butanedione monoxime) perturb specific cellular processes not explicitly related to the cell wall. Our results show that deficiencies in septation lead to fungi which are more susceptible to cell wall-perturbing compounds but are no more susceptible to other antifungal compounds than a control. This implies that septa play a critical role in surviving cell wall stress. IMPORTANCE The ability to compartmentalize potentially lethal damage via septation appears to provide filamentous fungi with a facile means to tolerate diverse forms of stress. However, it remains unknown whether this mechanism is deployed in response to all forms of stress or is limited to specific perturbations. Our results support the latter possibility by showing that presence of septa promotes survival in response to cell wall damage but plays no apparent role in coping with other unrelated forms of stress. Given that cell wall damage is a primary effect caused by exposure to the echinocandin class of antifungal agents, our results emphasize the important role that septa might play in enabling resistance to these drugs. Accordingly, the inhibition of septum formation could conceivably represent an attractive approach to potentiating the effects of echinocandins and mitigating resistance in human fungal pathogens.

Genetically stable CRISPR-based kill switches for engineered microbes.

Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development.

Multiplex Analysis to Unravel the Mode of Antifungal Activity of the Plant Defensin HsAFP1 in Single Yeast Cells.

Single cell analyses have gained increasing interest over bulk approaches because of considerable cell-to-cell variability within isogenic populations. Herein, flow cytometry remains golden standard due to its high-throughput efficiency and versatility, although it does not allow to investigate the interdependency of cellular events over time. Starting from our microfluidic platform that enables to trap and retain individual cells on a fixed location over time, here, we focused on unraveling kinetic responses of single Saccharomyces cerevisiae yeast cells upon treatment with the antifungal plant defensin HsAFP1. We monitored the time between production of reactive oxygen species (ROS) and membrane permeabilization (MP) in single yeast cells for different HsAFP1 doses using two fluorescent dyes with non-overlapping spectra. Within a time frame of 2 min, only <0.3% cells displayed time between the induction of ROS and MP. Reducing the time frame to 30 s did not result in increased numbers of cells with time between these events, pointing to ROS and MP induction as highly dynamic and correlated processes. In conclusion, using an in-house developed continuous microfluidic platform, we investigated the mode of action of HsAFP1 at single cell level, thereby uncovering the close interdependency between ROS induction and MP in yeast.