RpoN Regulates Virulence Factors of Pseudomonas aeruginosa via Modulating the PqsR Quorum Sensing Regulator

The alternative sigma factor RpoN regulates many cell functions, such as motility, quorum sensing, and virulence in the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). P. aeruginosa often evolves rpoN-negative variants during the chronic infection in cystic fibrosis patients. It is unclear how RpoN interacts with other regulatory mechanisms to control virulence of P. aeruginosa. In this study, we show that RpoN modulates the function of PqsR, a quorum sensing receptor regulating production of virulence factors including the phenazine pyocyanin. The ∆rpoN mutant is able to synthesize 4-quinolone signal molecule HHQ but unable to activate PqsR and Pseudomonas quinolone signal (pqs) quorum sensing. The ∆rpoN mutant produces minimal level of pyocyanin and is unable to produce the anti-staphylococcal agents. Providing pqsR in trans in the ∆rpoN mutant restores its pqs quorum sensing and virulence factor production to the wild-type level. Our study provides evidence that RpoN has a regulatory effect on P. aeruginosa virulence through modulating the function of the PqsR quorum sensing regulator.     

In Vitro Selection of Single-Stranded DNA Molecular Recognition Elements against S. aureus Alpha Toxin and Sensitive Detection in Human Serum

Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (K_d) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.     

Colonization and Infection of the Skin by S. aureus: Immune System Evasion and the Response to Cationic Antimicrobial Peptides

Staphylococcus aureus (S. aureus) is a widespread cutaneous pathogen responsible for the great majority of bacterial skin infections in humans. The incidence of skin infections by S. aureus reflects in part the competition between host cutaneous immune defenses and S. aureus virulence factors. As part of the innate immune system in the skin, cationic antimicrobial peptides (CAMPs) such as the β-defensins and cathelicidin contribute to host cutaneous defense, which prevents harmful microorganisms, like S. aureus, from crossing epithelial barriers. Conversely, S. aureus utilizes evasive mechanisms against host defenses to promote its colonization and infection of the skin. In this review, we focus on host-pathogen interactions during colonization and infection of the skin by S. aureus and methicillin-resistant Staphylococcus aureus (MRSA). We will discuss the peptides (defensins, cathelicidins, RNase7, dermcidin) and other mediators (toll-like receptor, IL-1 and IL-17) that comprise the host defense against S. aureus skin infection, as well as the various mechanisms by which S. aureus evades host defenses. It is anticipated that greater understanding of these mechanisms will enable development of more sustainable antimicrobial compounds and new therapeutic approaches to the treatment of S. aureus skin infection and colonization.     

Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.     

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)—an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a K_D value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure–activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.     

Functional Identification of Serine Hydroxymethyltransferase as a Key Gene Involved in Lysostaphin Resistance and Virulence Potential of Staphylococcus aureus Strains

Gaining an insight into the mechanism underlying antimicrobial-resistance development in Staphylococcus aureus is crucial for identifying effective antimicrobials. We isolated S. aureus sequence type 72 from a patient in whom the S. aureus infection was highly resistant to various antibiotics and lysostaphin, but no known resistance mechanisms could explain the mechanism of lysostaphin resistance. Genome-sequencing followed by subtractive and functional genomics revealed that serine hydroxymethyltransferase (glyA or shmT gene) plays a key role in lysostaphin resistance. Serine hydroxymethyltransferase (SHMT) is indispensable for the one-carbon metabolism of serine/glycine interconversion and is linked to folate metabolism. Functional studies revealed the involvement of SHMT in lysostaphin resistance, as ΔshmT was susceptible to the lysostaphin, while complementation of the knockout expressing shmT restored resistance against lysostaphin. In addition, the ΔshmT showed reduced virulence under in vitro (mammalian cell lines infection) and in vivo (wax-worm infection) models. The SHMT inhibitor, serine hydroxymethyltransferase inhibitor 1 (SHIN1), protected the 50% of the wax-worm infected with wild type S. aureus. These results suggest SHMT is relevant to the extreme susceptibility to lysostaphin and the host immune system. Thus, the current study established that SHMT plays a key role in lysostaphin resistance development and in determining the virulence potential of multiple drug-resistant S. aureus.     

Antimicrobial Resistance Profile of Planktonic and Biofilm Cells of Staphylococcus aureus and Coagulase-Negative Staphylococci

The objective of the present study was to determine the antimicrobial resistance profile of planktonic and biofilm cells of Staphylococcus aureus and coagulase-negative staphylococci (CoNS). Two hundred Staphylococcus spp. strains were studied, including 50 S. aureus and 150 CoNS strains (50 S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis, and 20 S. saprophyticus). Biofilm formation was investigated by adherence to polystyrene plates. Positive strains were submitted to the broth microdilution method to determine the minimum inhibitory concentration (MIC) for planktonic and biofilm cells and the minimal bactericidal concentration for biofilm cells (MBCB). Forty-nine Staphylococcus spp. strains (14 S. aureus, 13 S. epidermidis, 13 S. saprophyticus, 3 S. haemolyticus, 1 S. hominis, 3 S. warneri, and 2 S. lugdunensis) were biofilm producers. These isolates were evaluated regarding their resistance profile. Determination of planktonic cell MIC identified three (21.4%) S. aureus strains that were resistant to oxacillin and six (42.8%) that were resistant to erythromycin. Among the CoNS, 31 (88.6%) strains were resistant to oxacillin, 14 (40%) to erythromycin, 18 (51.4%) to gentamicin, and 8 (22.8%) to sulfamethoxazole/trimethoprim. None of the planktonic isolates were resistant to vancomycin or linezolid. MICs were 2-, 4-, 8-, and up to 16-fold higher for biofilm cells than for planktonic cells. This observation was more common for vancomycin and erythromycin. The MBCB ranged from 8 to >256 µg/mL for oxacillin, 128 to >128 µg/mL for vancomycin, 256 to >256 µg/mL for erythromycin and gentamicin, >64 µg/mL for linezolid, and 32/608 to >32/608 µg/mL for sulfamethoxazole/trimethoprim. The results showed considerably higher MICs for S. aureus and CoNS biofilm cells compared to planktonic cells. Analysis of MBCM confirmed that even high concentrations of vancomycin were unable to eliminate the biofilms of S. aureus and CoNS species. Linezolid was the most effective drug in inhibiting staphylococci in the biofilm, without an increase in the MIC, when compared to planktonic cells. None of the isolates were resistant to this drug.     

Immunogenicity and Cross-Protective Efficacy Induced by Outer Membrane Proteins from Salmonella Typhimurium Mutants with Truncated LPS in Mice

Lipopolysaccharide (LPS) is a major virulence factor present in the outer membrane of Salmonella enterica serovar Typhimurium (S. Typhimurium). Outer membrane proteins (OMPs) from Salmonella show high immunogenicity and provide protection against Salmonella infection, and truncated LPS alters the outer membrane composition of the cell wall. In our previous study, we demonstrated that Salmonella mutants carrying truncated LPS failed to induce strong immune responses and cross-reaction to other enteric bacteria, due to their high attenuation and low colonization in the host. Therefore, we plan to investigate whether outer membrane proteins from Salmonella mutants with truncated LPS resulting from a series of nonpolar mutations, including ∆waaC12, ∆waaF15, ∆waaG42, ∆rfaH49, ∆waaI43, ∆waaJ44, ∆waaL46, ∆wbaP45 and ∆wzy-48, affect immunogenicity and provide protection against diverse Salmonella challenge. In this study, the immunogenicity and cross-protection efficiency of purified OMPs from all mutants were investigated to explore a potential OMP vaccine to protect against homologous or heterologous serotype Salmonella challenge. The results demonstrated that OMPs from three Salmonella mutants (∆waaC12, ∆waaJ44 and ∆waaL46) induced higher immune responses and provided good protection against homologous S. Typhimurium. The OMPs from these three mutants were also selected to determine the cross-protective efficacy against homologous and heterologous serotype Salmonella. Our results indicated that the mutant ∆waaC12 can elicit higher cross-reactivity and can provide good protection against S. Choleraesuis and S. Enteritidis infection and that the cross-reactivity may be ascribed to an antigen of approximately 18.4–30 kDa.     

The Knockout of Enterobactin-Related Gene in Pectobacterium atrosepticum Results in Reduced Stress Resistance and Virulence towards the Primed Plants

Siderophores produced by microorganisms to scavenge iron from the environment have been shown to contribute to virulence and/or stress resistance of some plant pathogenic bacteria. Phytopathogenic bacteria of Pectobacterium genus possess genes for the synthesis of siderophore enterobactin, which role in plant-pathogen interactions has not been elucidated. In the present study we characterized the phenotype of the mutant strain of Pba deficient for the enterobactin-biosynthetic gene entA. We showed that enterobactin may be considered as a conditionally beneficial virulence factor of Pba. The entA knockout did not reduce Pba virulence on non-primed plants; however, salicylic acid-primed plants were more resistant to ΔentA mutant than to the wild type Pba. The reduced virulence of ΔentA mutant towards the primed plants is likely explained by its compromised resistance to oxidative stress.     

Silver Nanoparticles from Oregano Leaves’ Extracts as Antimicrobial Components for Non-Infected Hydrogel Contact Lenses

The oregano leaves’ extract (ORLE) was used for the formation of silver nanoparticles (AgNPs(ORLE)). ORLE and AgNPs(ORLE) (2 mg/mL) were dispersed in polymer hydrogels to give the pHEMA@ORLE_2 and pHEMA@AgNPs(ORLE)_2 using hydroxyethyl–methacrylate (HEMA). The materials were characterized by X-ray fluorescence (XRF) spectroscopy, X-ray powder diffraction analysis (XRPD), thermogravimetric differential thermal analysis (TG-DTA), derivative thermogravimetry/differential scanning calorimetry (DTG/DSC), ultraviolet (UV-Vis), and attenuated total reflection mode (ATR-FTIR) spectroscopies in solid state and UV–Vis in solution. The crystallite size value, analyzed with XRPD, was determined at 20 nm. The antimicrobial activity of the materials was investigated against Gram-negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The Gram-positive ones of the genus of Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) are known to be involved in microbial keratitis by the means of inhibitory zone (IZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The IZs, which developed upon incubation of P. aeruginosa, E. coli, S. epidermidis, and S. aureus with paper discs soaked in 2 mg/mL of AgNPs(ORLE), were 11.7 ± 0.7, 13.5 ± 1.9, 12.7 ± 1.7, and 14.3 ± 1.7 mm. When the same dose of ORLE was administrated, the IZs were 10.2 ± 0.7, 9.2 ± 0.5, 9.0 ± 0.0, and 9.0 ± 0.0 mm. The percent of bacterial viability when they were incubated over the polymeric hydrogel discs of pHEMA@AgNPs(ORLE)_2 was interestingly low (66.5, 88.3, 77.7, and 59.6%, respectively, against of P. aeruginosa, E. coli, S. epidermidis, and S. aureus) and those of pHEMA@ORLE_2 were 89.3, 88.1, 92.8, and 84.6%, respectively. Consequently, pHEMA@AgNPs(ORLE)_2 could be an efficient candidate toward the development of non-infectious contact lenses.     

Within-Host Adaptation of Staphylococcus aureus in a Bovine Mastitis Infection Is Associated with Increased Cytotoxicity

Within-host adaptation is a typical feature of chronic, persistent Staphylococcus aureus infections. Research projects addressing adaptive changes due to bacterial in-host evolution increase our understanding of the pathogen’s strategies to survive and persist for a long time in various hosts such as human and bovine. In this study, we investigated the adaptive processes of S. aureus during chronic, persistent bovine mastitis using a previously isolated isogenic strain pair from a dairy cow with chronic, subclinical mastitis, in which the last variant (host-adapted, Sigma factor SigB-deficient) quickly replaced the initial, dominant variant. The strain pair was cultivated under specific in vitro infection-relevant growth-limiting conditions (iron-depleted RPMI under oxygen limitation). We used a combinatory approach of surfaceomics, molecular spectroscopic fingerprinting and in vitro phenotypic assays. Cellular cytotoxicity assays using red blood cells and bovine mammary epithelial cells (MAC-T) revealed changes towards a more cytotoxic phenotype in the host-adapted isolate with an increased alpha-hemolysin (α-toxin) secretion, suggesting an improved capacity to penetrate and disseminate the udder tissue. Our results foster the hypothesis that within-host evolved SigB-deficiency favours extracellular persistence in S. aureus infections. Here, we provide new insights into one possible adaptive strategy employed by S. aureus during chronic, bovine mastitis, and we emphasise the need to analyse genotype–phenotype associations under different infection-relevant growth conditions.     

Multi-Omics Analysis Reveals Anti-Staphylococcus aureus Activity of Actinomycin D Originating from Streptomyces parvulus

Staphylococcus aureus (S. aureus) is a common pathogen that causes various serious diseases, including chronic infections. Discovering new antibacterial agents is an important aspect of the pharmaceutical field because of the lack of effective antibacterial drugs. In our research, we found that one anti-S. aureus substance is actinomycin D, originating from Streptomyces parvulus (S. parvulus); then, we further focused on the anti-S. aureus ability and the omics profile of S. aureus in response to actinomycin D. The results revealed that actinomycin D had a significant inhibitory activity on S. aureus with a minimum inhibitory concentration (MIC) of 2 μg/mL and a minimum bactericidal concentration (MBC) of 64 μg/mL. Bacterial reactive oxygen species (ROS) increased 3.5-fold upon treatment with actinomycin D, as was measured with the oxidation-sensitive fluorescent probe DCFH-DA, and H₂O₂ increased 3.5 times with treatment by actinomycin D. Proteomics and metabolomics, respectively, identified differentially expressed proteins in control and treatment groups, and the co-mapped correlation network of proteomics and metabolomics annotated five major pathways that were potentially related to disrupting the energy metabolism and oxidative stress of S. aureus. All findings contributed to providing new insight into the mechanisms of the anti-S. aureus effects of actinomycin D originating from S. parvulus.     

Polyphenylglyoxamide-Based Amphiphilic Small Molecular Peptidomimetics as Antibacterial Agents with Anti-Biofilm Activity

The rapid emergence of drug-resistant bacteria is a major global health concern. Antimicrobial peptides (AMPs) and peptidomimetics have arisen as a new class of antibacterial agents in recent years in an attempt to overcome antibiotic resistance. A library of phenylglyoxamide-based small molecular peptidomimetics was synthesised by incorporating an N-alkylsulfonyl hydrophobic group with varying alkyl chain lengths and a hydrophilic cationic group into a glyoxamide core appended to phenyl ring systems. The quaternary ammonium iodide salts 16d and 17c showed excellent minimum inhibitory concentration (MIC) of 4 and 8 μM (2.9 and 5.6 μg/mL) against Staphylococcus aureus, respectively, while the guanidinium hydrochloride salt 34a showed an MIC of 16 μM (8.5 μg/mL) against Escherichia coli. Additionally, the quaternary ammonium iodide salt 17c inhibited 70% S. aureus biofilm formation at 16 μM. It also disrupted 44% of pre-established S. aureus biofilms at 32 μM and 28% of pre-established E. coli biofilms 64 μM, respectively. A cytoplasmic membrane permeability study indicated that the synthesised peptidomimetics acted via disruption and depolarisation of membranes. Moreover, the quaternary ammonium iodide salts 16d and 17c were non-toxic against human cells at their therapeutic dosages against S. aureus.     

Recontamination of Healthcare Surfaces by Repeated Wiping with Biocide-Loaded Wipes: “One Wipe,One Surface,One Direction,Dispose” as Best Practice in the Clinical Environment

The wiping of high-touch healthcare surfaces made of metals, ceramics and plastics to remove bacteria is an accepted tool in combatting the transmission of healthcare-associated infections (HCAIs). In practice, surfaces may be repeatedly wiped using a single wipe, and the potential for recontamination may be affected by various factors. Accordingly, we studied how the surface to be wiped, the type of fibre in the wipe and how the presence of liquid biocide affected the degree of recontamination. Experiments were conducted using metal, ceramic and plastic healthcare surfaces, and two different wipe compositions (hygroscopic and hydrophilic), with and without liquid biocide. Despite initially high removal efficiencies of >70% during initial wiping, all healthcare surfaces were recontaminated with E. coli, S. aureus and E. faecalis when wiped more than once using the same wipe. Recontamination occurred regardless of the fibre composition of the wipe or the presence of a liquid biocide. The extent of recontamination by E. coli, S. aureus and E. faecalis bacteria also increased when metal healthcare surfaces possessed a higher microscale roughness (<1 μm), as determined by Atomic Force Microscopy (AFM). The high propensity for healthcare surfaces to be re-contaminated following initial wiping suggests that a “One wipe, One surface, One direction, Dispose” policy should be implemented and rigorously enforced.     

Perinatal Gram-Positive Bacteria Exposure Elicits Distinct Cytokine Responses In Vitro

During pregnancy, infections caused by the gram-positive bacteria Enterococcus faecalis (E. faecalis), Streptococcus agalacticae (S. agalacticae), and Staphylococcus aureus (S. aureus) are major reasons for preterm labor, neonatal prematurity, meningitis, or sepsis. Here, we propose cytokine responses to bacterial infections by the immature perinatal immune system as central players in the pathogenesis of preterm birth and neonatal sepsis. We aimed to close the gap in knowledge about such cytokine responses by stimulating freshly isolated umbilical blood mononuclear cells (UBMC) with lysates of E. faecalis, S. agalacticae, and S. aureus collected from pregnant women in preterm labor. Bacterial lysates and, principally, S. aureus and S. agalacticae distinctly triggered most of the eleven inflammatory, anti-inflammatory, TH₁/TH₂ cytokines, and chemokines quantified in UBMC culture media. Chemokines depicted the most robust induction. Among them, MIP-1β was further enhanced in UBMC from female compered to male newborn infants. Due to its stability and high levels, we investigated the diagnostic value of IL-8. IL-8 was critically upregulated in cord blood of preterm neonates suffering from infections compared to gestational age-matched controls. Our results provide novel clues about perinatal immunity, underscoring a potential value of IL-8 for the timely detection of infections and suggesting that MIP-1β constitutes an early determinant of sex-specific immunity, which may contribute, e.g., to male’s vulnerability to preterm birth.     

The Antimicrobial Peptide Temporin G: Anti-Biofilm,Anti-Persister Activities,and Potentiator Effect of Tobramycin Efficacy Against Staphylococcus aureus

Bacterial biofilms are a serious threat for human health, and the Gram-positive bacterium Staphylococcus aureus is one of the microorganisms that can easily switch from a planktonic to a sessile lifestyle, providing protection from a large variety of adverse environmental conditions. Dormant non-dividing cells with low metabolic activity, named persisters, are tolerant to antibiotic treatment and are the principal cause of recalcitrant and resistant infections, including skin infections. Antimicrobial peptides (AMPs) hold promise as new anti-infective agents to treat such infections. Here for the first time, we investigated the activity of the frog-skin AMP temporin G (TG) against preformed S. aureus biofilm including persisters, as well as its efficacy in combination with tobramycin, in inhibiting S. aureus growth. TG was found to provoke ~50 to 100% reduction of biofilm viability in the concentration range from 12.5 to 100 µM vs ATCC and clinical isolates and to be active against persister cells (about 70–80% killing at 50–100 µM). Notably, sub-inhibitory concentrations of TG in combination with tobramycin were able to significantly reduce S. aureus growth, potentiating the antibiotic power. No critical cytotoxicity was detected when TG was tested in vitro up to 100 µM against human keratinocytes, confirming its safety profile for the development of a new potential anti-infective drug, especially for treatment of bacterial skin infections.     

Investigating Pathogenicity and Virulence of Staphylococcus pettenkoferi: An Emerging Pathogen

Staphylococcus pettenkoferi is a coagulase-negative Staphylococcus identified in 2002 that has been implicated in human diseases as an opportunistic pathogenic bacterium. Its multiresistant character is becoming a major health problem, yet the pathogenicity of S. pettenkoferi is poorly characterized. In this study, the pathogenicity of a S. pettenkoferi clinical isolate from diabetic foot osteomyelitis was compared with a Staphylococcus aureus strain in various in vitro and in vivo experiments. Growth kinetics were compared against S. aureus, and bacteria survival was assessed in the RAW 264.7 murine macrophage cell line, the THP-1 human leukemia monocytic cell line, and the HaCaT human keratinocyte cell line. Ex vivo analysis was performed in whole blood survival assays and in vivo assays via the infection model of zebrafish embryos. Moreover, whole-genome analysis was performed. Our results show that S. pettenkoferi was able to survive in human blood, human keratinocytes, murine macrophages, and human macrophages. S. pettenkoferi demonstrated its virulence by causing substantial embryo mortality in the zebrafish model. Genomic analysis revealed virulence factors such as biofilm-encoding genes (e.g., icaABCD; rsbUVW) and regulator-encoding genes (e.g., agr, mgrA, sarA, saeS) well characterized in S. aureus. This study thus advances the knowledge of this under-investigated pathogen and validates the zebrafish infection model for this bacterium.     

Functionalization of Crosslinked Sodium Alginate/Gelatin Wet-Spun Porous Fibers with Nisin Z for the Inhibition of Staphylococcus aureus-Induced Infections

Nisin Z, an amphipathic peptide, with a significant antibacterial activity against Gram-positive bacteria and low toxicity in humans, has been studied for food preservation applications. Thus far, very little research has been done to explore its potential in biomedicine. Here, we report the modification of sodium alginate (SA) and gelatin (GN) blended microfibers, produced via the wet-spinning technique, with Nisin Z, with the purpose of eradicating Staphylococcus aureus-induced infections. Wet-spun SAGN microfibers were successfully produced at a 70/30% v/v of SA (2 wt%)/GN (1 wt%) polymer ratio by extrusion within a calcium chloride (CaCl₂) coagulation bath. Modifications to the biodegradable fibers’ chemical stability and structure were then introduced via crosslinking with CaCl₂ and glutaraldehyde (SAGNCL). Regardless of the chemical modification employed, all microfibers were labelled as homogeneous both in size (≈246.79 µm) and shape (cylindrical and defect-free). SA-free microfibers, with an increased surface area for peptide immobilization, originated from the action of phosphate buffer saline solution on SAGN fibers, were also produced (GNCL). Their durability in physiological conditions (simulated body fluid) was, however, compromised very early in the experiment (day 1 and 3, with and without Nisin Z, respectively). Only the crosslinked SAGNCL fibers remained intact for the 28 day-testing period. Their thermal resilience in comparison with the unmodified and SA-free fibers was also demonstrated. Nisin Z was functionalized onto the unmodified and chemically altered fibers at an average concentration of 178 µg/mL. Nisin Z did not impact on the fiber’s morphology nor on their chemical/thermal stability. However, the peptide improved the SA fibers (control) structural integrity, guaranteeing its stability for longer, in physiological conditions. Its main effect was detected on the time-kill kinetics of the bacteria S. aureus. SAGNCL and GNCL loaded with Nisin Z were capable of progressively eliminating the bacteria, reaching an inhibition superior to 99% after 24 h of culture. The peptide-modified SA and SAGN were not as effective, losing their antimicrobial action after 6 h of incubation. Bacteria elimination was consistent with the release kinetics of Nisin Z from the fibers. In general, data revealed the increased potential and durable effect of Nisin Z (significantly superior to its free, unloaded form) against S. aureus-induced infections, while loaded onto prospective biomedical wet-spun scaffolds.     

Bitter Taste Receptor T2R14 Modulates Gram-Positive Bacterial Internalization and Survival in Gingival Epithelial Cells

Bitter-taste receptors (T2Rs) have emerged as key players in host–pathogen interactions and important modulators of oral innate immunity. Previously, we reported that T2R14 is expressed in gingival epithelial cells (GECs) and interacts with competence stimulating peptides (CSPs) secreted by the cariogenic Streptococcus mutans. The underlying mechanisms of the innate immune responses and physiological effects of T2R14 on Gram-positive bacteria are not well characterized. In this study, we examined the role of T2R14 in internalization and growth inhibitory effects on Gram-positive bacteria, namely Staphylococcus aureus and S. mutans. We utilized CRISPR-Cas9 T2R14 knockdown (KD) GECs as the study model to address these key physiological mechanisms. Our data reveal that the internalization of S. aureus is significantly decreased, while the internalization of S. mutans remains unaffected upon knockdown of T2R14 in GECs. Surprisingly, GECs primed with S. mutans CSP-1 resulted in an inhibition of growth for S. aureus, but not for S. mutans. The GECs infected with S. aureus induced T2R14-dependent human β-defensin-2 (hBD-2) secretion; however, S. mutans–infected GECs did not induce hBD-2 secretion, but induced T2R14 dependent IL-8 secretion. Interestingly, our results show that T2R14 KD affects the cytoskeletal reorganization in GECs, thereby inhibiting S. aureus internalization. Our study highlights the distinct mechanisms and a direct role of T2R14 in influencing physiological responses to Gram-positive bacteria in the oral cavity.     

Composition,Cytotoxic and Antimicrobial Activities of Satureja intermedia C.A.Mey Essential Oil

In this study, the essential oil (EO) constituents from the aerial parts of Satureja intermedia C.A.Mey were detected by GC and GC/MS. The antimicrobial activity of EO on oral pathogens and its cytotoxicity to human cancer cells were determined by the microbroth dilution method and the crystal violet staining method, respectively. Thirty-nine compounds were identified and the main EO constituents were γ-terpinene (37.1%), thymol (30.2%), p-cymene (16.2%), limonene (3.9%), α-terpinene (3.3%), myrcene (2.5%), germacrene B (1.4%), elemicine (1.1%) and carvacrol (0.5%). The S. intermedia EO showed a concentration-dependent decrease in viability of Hep-G2 (hepatocellular carcinoma) and MCF-7 (breast adenocarcinoma) human cancer cell lines (p < 0.05). Antimicrobial screening of S. intermedia EO demonstrated slight antibacterial and antifungal activities against Streptococcus mutants, S. salivarius, Enterococcus faecalis, Staphylococcus aureus, Candida albicans and C. glabrata. Further preclinical studies are needed to assess the efficacy and safety of S. intermedia EO as a new promising anticancer agent.