Cows with diverging haplotypes show differences in differential milk cell count, milk parameters and vaginal temperature after S. aureus challenge but not after E. coli challenge.

BACKGROUND: In dairy cattle, mastitis causes high financial losses and impairs animal well-being. Genetic selection is used to breed cows with reduced mastitis susceptibility. Techniques such as milk cell flow cytometry may improve early mastitis diagnosis. In a highly standardized in vivo infection model, 36 half-sib cows were selected for divergent paternal Bos taurus chromosome 18 haplotypes (Q vs. q) and challenged with Escherichia coli for 24 h or Staphylococcus aureus for 96 h, after which the samples were analyzed at 12 h intervals. Vaginal temperature (VT) was recorded every three minutes. The objective of this study was to compare the differential milk cell count (DMCC), milk parameters (fat %, protein %, lactose %, pH) and VT between favorable (Q) and unfavorable (q) haplotype cows using Bayesian models to evaluate their potential as improved early indicators of differential susceptibility to mastitis. RESULTS: After S. aureus challenge, compared to the Q half-sibship cows, the milk of the q cows exhibited higher PMN levels according to the DMCC (24 h, p < 0.001), a higher SCC (24 h, p < 0.01 and 36 h, p < 0.05), large cells (24 h, p < 0.05) and more dead (36 h, p < 0.001) and live cells (24 h, p < 0.01). The protein % was greater in Q milk than in q milk at 0 h (p = 0.025). In the S. aureus group, Q cows had a greater protein % (60 h, p = 0.048) and fat % (84 h, p = 0.022) than q cows. Initially, the greater VT of S. aureus-challenged q cows (0 and 12-24 h, p < 0.05) reversed to a lower VT in q cows than in Q cows (48-60 h, p < 0.05). Additionally, the following findings emphasized the validity of the model: in the S. aureus group all DMCC subpopulations (24 h-96 h, p < 0.001) and in the E. coli group nearly all DMCC subpopulations (12 h-24 h, p < 0.001) were higher in challenged quarters than in unchallenged quarters. The lactose % was lower in the milk samples of E. coli-challenged quarters than in those of S. aureus-challenged quarters (24 h, p < 0.001). Between 12 and 18 h, the VT was greater in cows challenged with E. coli than in those challenged with S. aureus (3-h interval approach, p < 0.001). CONCLUSION: This in vivo infection model confirmed specific differences between Q and q cows with respect to the DMCC, milk component analysis results and VT results after S. aureus inoculation but not after E. coli challenge. However, compared with conventional milk cell analysis monitoring, e.g., the global SCC, the DMCC analysis did not provide refined phenotyping of the pathogen response.

Staphylococcus Aureus Membrane Vesicles Kill Tumor Cells Through a Caspase-1-Dependent Pyroptosis Pathway.

Introduction: Nanosized outer membrane vesicles (OMVs) from Gram-negative bacteria have attracted increasing interest because of their antitumor activity. However, the antitumor effects of MVs isolated from Gram-positive bacteria have rarely been investigated. Methods: MVs of Staphylococcus aureus USA300 were prepared and their antitumor efficacy was evaluated using tumor-bearing mouse models. A gene knock-in assay was performed to generate luciferase Antares2-MVs for bioluminescent detection. Cell counting kit-8 and lactic dehydrogenase release assays were used to detect the toxicity of the MVs against tumor cells in vitro. Active caspase-1 and gasdermin D (GSDMD) levels were determined using Western blot, and the tumor inhibition ability of MVs was determined in B16F10 cells treated with a caspase-1 inhibitor. Results: The vesicular particles of S. aureus USA300 MVs were 55.23 ± 8.17 nm in diameter, and 5 µg of MVs remarkably inhibited the growth of B16F10 melanoma in C57BL/6 mice and CT26 colon adenocarcinoma in BALB/c mice. The bioluminescent signals correlated well with the concentrations of the engineered Antares2-MVs (R2 = 0.999), and the sensitivity for bioluminescence imaging was 4 × 10-3 µg. Antares2-MVs can directly target tumor tissues in vivo, and 20 µg/mL Antares2-MVs considerably reduced the growth of B16F10 and CT26 tumor cells, but not non-carcinomatous bEnd.3 cells. MV treatment substantially increased the level of active caspase-1, which processes GSDMD to trigger pyroptosis in tumor cells. Blocking caspase-1 activation with VX-765 significantly protected tumor cells from MV killing in vitro and in vivo. Conclusion: S. aureus MVs can kill tumor cells by activating the pyroptosis pathway, and the induction of pyroptosis in tumor cells is a promising strategy for cancer treatment.

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

Hindering the biofilm of microbial pathogens and cancer cell lines development using silver nanoparticles synthesized by epidermal mucus proteins from Clarias gariepinus.

Scientists know very little about the mechanisms underlying fish skin mucus, despite the fact that it is a component of the immune system. Fish skin mucus is an important component of defence against invasive infections. Recently, Fish skin and its mucus are gaining interest among immunologists. Characterization was done on the obtained silver nanoparticles Ag combined with Clarias gariepinus catfish epidermal mucus proteins (EMP-Ag-NPs) through UV-vis, FTIR, XRD, TEM, and SEM. Ag-NPs ranged in size from 4 to 20 nm, spherical in form and the angles were 38.10°, 44.20°, 64.40°, and 77.20°, Where wavelength change after formation of EMP-Ag-NPs as indicate of dark brown, the broad band recorded at wavelength at 391 nm. Additionally, the antimicrobial, antibiofilm and anticancer activities of EMP-Ag-NPs was assessed. The present results demonstrate high activity against unicellular fungi C. albicans, followed by E. faecalis. Antibiofilm results showed strong activity against both S. aureus and P. aeruginosa pathogens in a dose-dependent manner, without affecting planktonic cell growth. Also, cytotoxicity effect was investigated against normal cells (Vero), breast cancer cells (Mcf7) and hepatic carcinoma (HepG2) cell lines at concentrations (200-6.25 µg/mL) and current results showed highly anticancer effect of Ag-NPs at concentrations 100, 5 and 25 µg/mL exhibited rounding, shrinkage, deformation and granulation of Mcf7 and HepG2 with IC50 19.34 and 31.16 µg/mL respectively while Vero cells appeared rounded at concentration 50 µg/mL and normal shape at concentration 25, 12.5 and 6.25 µg/ml with IC50 35.85 µg/mL. This study evidence the potential efficacy of biologically generated Ag-NPs as a substitute medicinal agent against harmful microorganisms. Furthermore, it highlights their inhibitory effect on cancer cell lines.

Unveiling antibiofilm potential: proteins from Priestia sp. targeting Staphylococcus aureus biofilm formation.

Staphylococcus aureus is the etiologic agent of many nosocomial infections, and its biofilm is frequently isolated from medical devices. Moreover, the dissemination of multidrug-resistant (MDR) strains from this pathogen, such as methicillin-resistant S. aureus (MRSA) strains, is a worldwide public health issue. The inhibition of biofilm formation can be used as a strategy to weaken bacterial resistance. Taking that into account, we analysed the ability of marine sponge-associated bacteria to produce antibiofilm molecules, and we found that marine Priestia sp., isolated from marine sponge Scopalina sp. collected on the Brazilian coast, secretes proteins that impair biofilm development from S. aureus. Partially purified proteins (PPP) secreted after 24 hours of bacterial growth promoted a 92% biofilm mass reduction and 4.0 µg/dL was the minimum concentration to significantly inhibit biofilm formation. This reduction was visually confirmed by light microscopy and Scanning Electron Microscopy (SEM). Furthermore, biochemical assays showed that the antibiofilm activity of PPP was reduced by ethylenediaminetetraacetic acid (EDTA) and 1,10 phenanthroline (PHEN), while it was stimulated by zinc ions, suggesting an active metallopeptidase in PPP. This result agrees with mass spectrometry (MS) identification, which indicated the presence of a metallopeptidase from the M28 family. Additionally, whole-genome sequencing analysis of Priestia sp. shows that gene ywad, a metallopeptidase-encoding gene, was present. Therefore, the results presented herein indicate that PPP secreted by the marine Priestia sp. can be explored as a potential antibiofilm agent and help to treat chronic infections.

Determining the Young's Modulus of the Bacterial Cell Envelope.

Bacteria experience substantial physical forces in their natural environment, including forces caused by osmotic pressure, growth in constrained spaces, and fluid shear. The cell envelope is the primary load-carrying structure of bacteria, but the mechanical properties of the cell envelope are poorly understood; reports of Young's modulus of the cell envelope of Escherichia coli range from 2 to 18 MPa. We developed a microfluidic system to apply mechanical loads to hundreds of bacteria at once and demonstrated the utility of the approach for evaluating whole-cell stiffness. Here, we extend this technique to determine Young's modulus of the cell envelope of E. coli and of the pathogens Vibrio cholerae and Staphylococcus aureus. An optimization-based inverse finite element analysis was used to determine the cell envelope Young's modulus from observed deformations. The Young's modulus values of the cell envelope were 2.06 ± 0.04 MPa for E. coli, 0.84 ± 0.02 MPa for E. coli treated with a chemical (A22) known to reduce cell stiffness, 0.12 ± 0.03 MPa for V. cholerae, and 1.52 ± 0.06 MPa for S. aureus (mean ± SD). The microfluidic approach allows examination of hundreds of cells at once and is readily applied to Gram-negative and Gram-positive organisms as well as rod-shaped and cocci cells, allowing further examination of the structural causes behind differences in cell envelope Young's modulus among bacterial species and strains.

CXCR2 perturbation promotes Staphylococcus aureus implant-associated infection.

Introduction. Staphylococcus aureus is the leading cause of acute medical implant infections, representing a significant modern medical concern. The success of S. aureus as a pathogen in these cases resides in its arsenal of virulence factors, resistance to multiple antimicrobials, mechanisms of immune modulation, and ability to rapidly form biofilms associated with implant surfaces. S. aureus device-associated, biofilm-mediated infections are often persistent and notoriously difficult to treat, skewing innate immune responses to promote chronic reoccurring infections. While relatively little is known of the role neutrophils play in response to acute S. aureus biofilm infections, these effector cells must be efficiently recruited to sites of infection via directed chemotaxis. Here we investigate the effects of modulating CXC chemokine receptor 2 (CXCR2) activity, predominantly expressed on neutrophils, during S. aureus implant-associated infection.Hypothesis. We hypothesize that modulation of CXCR2 expression and/or signalling activities during S. aureus infection, and thus neutrophil recruitment, extravasation and antimicrobial activity, will affect infection control and bacterial burdens in a mouse model of implant-associated infection.Aim. This investigation aims to elucidate the impact of altered CXCR2 activity during S. aureus biofilm-mediated infection that may help develop a framework for an effective novel strategy to prevent morbidity and mortality associated with implant infections.Methodology. To examine the role of CXCR2 during S. aureus implant infection, we employed a mouse model of indwelling subcutaneous catheter infection using a community-associated methicillin-resistant S. aureus (MRSA) strain. To assess the role of CXCR2 induction or inhibition during infection, treatment groups received daily intraperitoneal doses of either Lipocalin-2 (Lcn2) or AZD5069, respectively. At the end of the study, catheters and surrounding soft tissues were analysed for bacterial burdens and dissemination, and Cxcr2 transcription within the implant-associated tissues was quantified.Results. Mice treated with Lcn2 developed higher bacterial burdens within the soft tissue surrounding the implant site, which was associated with increased Cxcr2 expression. AZD5069 treatment also resulted in increased implant- and tissues-associated bacterial titres, as well as enhanced Cxcr2 expression.Conclusion. Our results demonstrate that CXCR2 plays an essential role in regulating the severity of S. aureus implant-associated infections. Interestingly, however, perturbation of CXCR2 expression or signalling both resulted in enhanced Cxcr2 transcription and elevated implant-associated bacterial burdens. Thus, CXCR2 appears finely tuned to efficiently recruit effector cells and mediate control of S. aureus biofilm-mediated infection.

PDADMAC/Alginate-Coated Gold Nanorod For Eradication of Staphylococcus Aureus Biofilms.

Introduction: Over 75% of clinical microbiological infections are caused by bacterial biofilms that grow on wounds or implantable medical devices. This work describes the development of a new poly(diallyldimethylammonium chloride) (PDADMAC)/alginate-coated gold nanorod (GNR/Alg/PDADMAC) that effectively disintegrates the biofilms of Staphylococcus aureus (S. aureus), a prominent pathogen responsible for hospital-acquired infections. Methods: GNR was synthesised via seed-mediated growth method, and the resulting nanoparticles were coated first with Alg and then PDADMAC. FTIR, zeta potential, transmission electron microscopy, and UV-Vis spectrophotometry analysis were performed to characterise the nanoparticles. The efficacy and speed of the non-coated GNR and GNR/Alg/PDADMAC in disintegrating S. aureus-preformed biofilms, as well as their in vitro biocompatibility (L929 murine fibroblast) were then studied. Results: The synthesised GNR/Alg/PDADMAC (mean length: 55.71 ± 1.15 nm, mean width: 23.70 ± 1.13 nm, aspect ratio: 2.35) was biocompatible and potent in eradicating preformed biofilms of methicillin-resistant (MRSA) and methicillin-susceptible S. aureus (MSSA) when compared to triclosan, an antiseptic used for disinfecting S. aureus colonisation on abiotic surfaces in the hospital. The minimum biofilm eradication concentrations of GNR/Alg/PDADMAC (MBEC50 for MRSA biofilm = 0.029 nM; MBEC50 for MSSA biofilm = 0.032 nM) were significantly lower than those of triclosan (MBEC50 for MRSA biofilm = 10,784 nM; MBEC50 for MRSA biofilm 5967 nM). Moreover, GNR/Alg/PDADMAC was effective in eradicating 50% of MRSA and MSSA biofilms within 17 min when used at a low concentration (0.15 nM), similar to triclosan at a much higher concentration (50 µM). Disintegration of MRSA and MSSA biofilms was confirmed by field emission scanning electron microscopy and confocal laser scanning microscopy. Conclusion: These findings support the potential application of GNR/Alg/PDADMAC as an alternative agent to conventional antiseptics and antibiotics for the eradication of medically important MRSA and MSSA biofilms.

Exploration of agr types, virulence-associated genes, and biofilm formation ability in Staphylococcus aureus isolates from hemodialysis patients with vascular access infections.

Introduction: Staphylococcus aureus, is a pathogen commonly encountered in both community and hospital settings. Patients receiving hemodialysis treatment face an elevated risk of vascular access infections (VAIs) particularly Staphylococcus aureus, infection. This heightened risk is attributed to the characteristics of Staphylococcus aureus, , enabling it to adhere to suitable surfaces and form biofilms, thereby rendering it resistant to external interventions and complicating treatment efforts. Methods: Therefore this study utilized PCR and microtiter dish biofilm formation assay to determine the difference in the virulence genes and biofilm formation among in our study collected of 103 Staphylococcus aureus, isolates from hemodialysis patients utilizing arteriovenous grafts (AVGs), tunneled cuffed catheters (TCCs), and arteriovenous fistulas (AVFs) during November 2013 to December 2021. Results: Our findings revealed that both MRSA and MSSA isolates exhibited strong biofilm production capabilities. Additionally, we confirmed the presence of agr types and virulence genes through PCR analysis. The majority of the collected isolates were identified as agr type I. However, agr type II isolates displayed a higher average number of virulence genes, with MRSA isolates exhibiting a variety of virulence genes. Notably, combinations of biofilm-associated genes, such as eno-clfA-clfB-fib-icaA-icaD and eno-clfA-clfB-fib-fnbB-icaA-icaD, were prevalent among Staphylococcus aureus, isolates obtained from vascular access infections. Discussion: These insights contribute to a better understanding of the molecular characteristics associated with Staphylococcus aureus, infections in hemodialysis patients and provided more targeted and effective treatment approaches.

Evaluation of surface roughness, wettability and adhesion of multispecies biofilm on 3D-printed resins for the base and teeth of complete dentures.

OBJECTIVE: This study evaluated the surface roughness, wettability and adhesion of multispecies biofilms (Candida albicans, Staphylococcus aureus and Streptococcus mutans) on 3D-printed resins for complete denture bases and teeth compared to conventional resins (heat-polymerized acrylic resin; artificial pre-fabricated teeth). METHODOLOGY: Circular specimens (n=39; 6.0 mm Ø × 2.0 mm) of each group were subjected to roughness (n=30), wettability (n=30) and biofilm adhesion (n=9) tests. Three roughness measurements were taken by laser confocal microscopy and a mean value was calculated. Wettability was evaluated by the contact angle of sessile drop method, considering the mean of the three evaluations per specimen. In parallel, microorganism adhesion to resin surfaces was evaluated using a multispecies biofilm model. Microbial load was evaluated by determining the number of Colony Forming Units (CFU/mL) and by scanning electron microscopy (SEM). Data were subjected to the Wald test in a generalized linear model with multiple comparisons and Bonferroni adjustment, as well as two-way ANOVA (α=5%). RESULTS: The roughness of the conventional base resin (0.01±0.04) was lower than that of the conventional tooth (0.14±0.04) (p=0.023) and 3D-printed base (0.18±0.08) (p<0.001). For wettability, conventional resin (84.20±5.57) showed a higher contact angle than the 3D-printed resin (60.58±6.18) (p<0.001). Higher microbial loads of S. mutans (p=0.023) and S. aureus (p=0.010) were observed on the surface of the conventional resin (S. mutans: 5.48±1.55; S. aureus: 7.01±0.57) compared to the 3D-printed resin (S. mutans: 4.11±1.96; S. aureus: 6.42±0.78). The adhesion of C. albicans was not affected by surface characteristics. The conventional base resin showed less roughness than the conventional dental resin and the printed base resin. CONCLUSION: The 3D-printed resins for base and tooth showed less hydrophobicity and less adhesion of S. mutans and S. aureus than conventional resins.

Antibacterial and antibiofilm activities of iodinated hydrocarbons against Vibrio parahaemolyticus and Staphylococcus aureus.

Food-related illnesses have become a growing public concern due to their considerable socioeconomic and medical impacts. Vibrio parahaemolyticus and Staphylococcus aureus have been implicated as causative organisms of food-related infections and poisoning, and both can form biofilms which confer antibiotic resistance. Hence, the need for continuous search for compounds with antibiofilm and antivirulence properties. In this study, 22 iodinated hydrocarbons were screened for their antibiofilm activity, and of these, iodopropynyl butylcarbamate (IPBC) was found to effectively control biofilm formation of both pathogens with a MIC of 50 µg/mL which was bactericidal to V. parahaemolyticus and S. aureus. Microscopic studies confirmed IPBC inhibits biofilm formation of both bacteria and also disrupted their mixed biofilm formation. Furthermore, IPBC suppressed virulence activities such as motility and hemolytic activity of V. parahaemolyticus and the cell surface hydrophobicity of S. aureus. It exhibited a preservative potential against both pathogens in a shrimp model. IPBC disrupted the cell membrane of S. aureus and V. parahaemolyticus and differentially affected gene expressions related to biofilm formation and virulence. Additionally, it displayed broad-spectrum antibiofilm activities against other clinically relevant pathogens. These findings indicate IPBC offers a potential means of controlling infections mediated by Vibrio and Staphylococcus biofilms.

Adhesion of Klebsiella oxytoca to bladder or lung epithelial cells is promoted by the presence of other opportunistic pathogens.

The intestinal and respiratory tracts of healthy individuals serve as habitats for a diverse array of microorganisms, among which Klebsiella oxytoca holds significance as a causative agent in numerous community- and hospital-acquired infections, often manifesting in polymicrobial contexts. In specific circumstances, K. oxytoca, alongside other constituents of the gut microbiota, undergoes translocation to distinct physiological niches. In these new environments, it engages in close interactions with other microbial community members. As this interaction may progress to co-infection where the virulence of involved pathogens may be promoted and enhance disease severity, we investigated how K. oxytoca affects the adhesion of commonly co-isolated bacteria and vice versa during co-incubation of different biotic and abiotic surfaces. Co-incubation was beneficial for the adhesion of at least one of the two co-cultured strains. K. oxytoca enhanced the adhesion of other enterobacteria strains to polystyrene and adhered more efficiently to bladder or lung epithelial cell lines in the presence of most enterobacteria strains and S. aureus. This effect was accompanied by bacterial coaggregation mediated by carbohydrate-protein interactions occurring between bacteria. These interactions occur only in sessile, but not planktonic populations, and depend on the features of the surface. The data are of particular importance for the risk assessment of the urinary and respiratory tract infections caused by K. oxytoca, including those device-associated. In this paper, we present the first report on K. oxytoca ability to acquire increased adhesive capacities on epithelial cells through interactions with common causal agents of urinary and respiratory tract infections.

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.

Enhanced protein-metabolite correlation analysis: To investigate the association between Staphylococcus aureus mastitis and metabolic immune pathways.

Mastitis is a disease characterized by congestion, swelling, and inflammation of the mammary gland and usually caused by infection with pathogenic microorganisms. Furthermore, the development of mastitis is closely linked to the exogenous pathway of the gastrointestinal tract. However, the regulatory mechanisms governing the gut-metabolism-mammary axis remain incompletely understood. The present study revealed alterations in the gut microbiota of mastitis rats characterized by an increased abundance of the Proteobacteria phylum. Plasma analysis revealed significantly higher levels of L-isoleucine and cholic acid along with 7-ketodeoxycholic acid. Mammary tissue showed elevated levels of arachidonic acid metabolites and norlithocholic acid. Proteomic analysis showed increased levels of IFIH1, Tnfaip8l2, IRGM, and IRF5 in mastitis rats, which suggests that mastitis triggers an inflammatory response and immune stress. Follistatin (Fst) and progesterone receptor (Pgr) were significantly downregulated, raising the risk of breast cancer. Extracellular matrix (ECM) receptors and focal adhesion signaling pathways were downregulated, while blood-milk barrier integrity was disrupted. Analysis of protein-metabolic network regulation revealed that necroptosis, protein digestion and absorption, and arachidonic acid metabolism were the principal regulatory pathways involved in the development of mastitis. In short, the onset of mastitis leads to changes in the microbiota and alterations in the metabolic profiles of various biological samples, including colonic contents, plasma, and mammary tissue. Key manifestations include disturbances in bile acid metabolism, amino acid metabolism, and arachidonic acid metabolism. At the same time, the integrity of the blood-milk barrier is compromised while inflammation is promoted, thereby reducing cell adhesion in the mammary glands. These findings contribute to a more comprehensive understanding of the metabolic status of mastitis and provide new insights into its impact on the immune system.

Eliminating extracellular autoinducing peptide signals inhibits the Staphylococcus aureus quorum sensing agr system.

An accessory gene regulator (agr) in the quorum sensing (QS) system in Staphylococcus aureus contributes to host infection, virulence factor production, and resistance to oxidative damage. Artificially maintaining the inactive state of agr QS impedes the host infection strategy of S. aureus and inhibits toxin production. The QS system performs intercellular signal transduction, which is activated by the mature autoinducer peptide (AIP). It is released from cells after AgrD peptide processing as an intercellular signal associated with increased bacterial cell density. This study evaluated the effectiveness of inhibiting agr QS wherein AIP trap carriers were made to coexist when culturing Staphylococcus aureus. Immersing a nitrocellulose (NC) membrane in Staphylococcus aureus ATCC 12600 culture inhibited QS-dependent α-hemolysin production, which significantly reduced the hemolysis ratio of sheep red blood cells by the culture supernatant. A quartz crystal microbalance analysis supported AIP adsorption onto the NC membrane. Adding the NC membrane during culture was found to maintain the expression levels of the agr QS gene agrA and α-hemolysin gene hla lower than that when it was not added. Eliminating extracellular AIP signals allowed agr QS to remain inactive and prevented QS-dependent α-hemolysin expression. Isolating intercellular signals secreted outside the cell is an effective strategy to suppress gene expression in bacterial cells that collaborate via intercellular signaling.

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Staphylococcus aureus promotes its intracellular survival by inhibiting Rab11-Rab11FIP4-mediated vesicle trafficking.

Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.

The Staphylococcus aureus regulatory program in a human skin-like environment.

Staphylococcus aureus is a Gram-positive pathogen responsible for the majority of skin and soft tissue infections (SSTIs). S. aureus colonizes the anterior nares of approximately 20%-30% of the population and transiently colonizes the skin, thereby increasing the risk of developing SSTIs and more serious infections. Current laboratory models that mimic the skin surface environment are expensive, require substantial infrastructure, and limit the scope of bacterial physiology studies under human skin conditions. To overcome these limitations, we developed a cost-effective, open-source, chemically defined media recipe termed skin-like medium (SLM) that incorporates key aspects of the human skin surface environment and supports growth of several staphylococcal species. We utilized SLM to investigate the transcriptional response of methicillin-resistant Staphylococcus aureus (MRSA) following growth in SLM compared to a commonly used laboratory media. Through RNA-seq analysis, we observed the upregulation of several virulence factors, including genes encoding functions involved in adhesion, proteolysis, and cytotoxicity. To further explore these findings, we conducted quantitative reverse transcription-PCR (qRT-PCR) experiments to determine the influence of media composition, pH, and temperature on the transcriptional response of key factors involved in adhesion and virulence. We also demonstrated that MRSA primed in SLM adhered better to human corneocytes and demonstrated adhesin-specific phenotypes that previously required genetic manipulation. This improved adherence to corneocytes was dependent on both acidic pH and growth in SLM. These results support the potential utility of SLM as an in vitro model for assessing staphylococcal physiology and metabolism on human skin. IMPORTANCE: Staphylococcus aureus is the major cause of skin diseases, and its increased prevalence in skin colonization and infections present a need to understand its physiology in this environment. The work presented here outlines S. aureus upregulation of colonization and virulence factors using a newly developed medium that strives to replicate the human skin surface environment and demonstrates roles for adhesins clumping factor A (ClfA), serine-rich repeat glycoprotein adhesin (SraP), and the fibronectin binding proteins (Fnbps) in human corneocyte adherence.

A novel C-type lectin protein (BjCTL5) interacts with apoptosis stimulating proteins of p53 (ASPP) to activate NF-κB signaling pathway in primitive chordate.

C-type lectin proteins (CTLs), a group of pattern recognition receptors (PRRs), play pivotal roles in immune responses. However, the signal transduction and regulation of CTLs in cephalochordates have yet to be explored. In this study, we examined the composition of CTLs in Branchiostoma japonicum, identifying a total of 272 CTLs. These CTLs underwent further analysis concerning domain arrangement, tandem and segmental duplication events. A multidomain C-type lectin gene, designated as BjCTL5, encompassing CLECT, KR, CUB, MAM, and SR domains, was the focal point of our investigation. BjCTL5 exhibits ubiquitous expression across all detected tissues and is responsive to stimulation by LPS, mannose, and poly (I:C). The recombinant protein of BjCTL5 can bind to Escherichia coli and Staphylococcus aureus, inducing their agglutination and inhibiting the proliferation of S. aureus. Yeast two-hybrid, CoIP, and confocal immunofluorescence experiments revealed the interaction between BjCTL5 and apoptosis-stimulating proteins of p53, BjASPP. Intriguingly, BjCTL5 was observed to induce the luciferase activity of the NF-κB promoter in HEK293T cells. These results suggested a potential interaction between BjCTL5 and BjASPP, implicating that they involve in the activation of the NF-κB signaling pathway, which provides an evolutionary viewpoint on NF-κB signaling pathway in primitive chordate.

Preparation and Physicochemical Characterization of Hyaluronic Acid-Lysine Nanogels Containing Serratiopeptidase to Control Biofilm Formation.

Remarkable resistance of bacterial biofilms to high doses of antimicrobials and antibiotics is one of their main challenges. Encapsulation of proteolytic enzymes is one of the suggested strategies to tackle this problem. In this regard, the antibacterial and anti-biofilm activity of biocompatible hyaluronic acid- Lysine nanogels containing serratiopeptidase (SRP-loaded HA-Lys nanogel) was assessed against P. aeruginosa and S. aureus strains. SRP-loaded HA-Lys nanogel was prepared using dropping method and optimized by Box-Behnken experimental design. These formulations were studied for physical characterization, release profile, stability, bioactivity, and anti-biofilm effects. The particle size, polydispersity index (PDI), and surface charge were measured by Zetasizer Nano ZS. The average particle size and zeta potential of the optimum sample were 156 nm and -14.1 mV, respectively. SRP release showed an initial burst followed by sustained release and the highest release was around 77%. Enzyme biological activity data revealed the higher efficiency of free SRP compared to SRP-loaded HA-Lys nanogel. The time-kill assay showed that both forms of SRP-loaded HA-Lys nanogel and blank HA-Lys nanogel showed significant antimicrobial activity against examined bacteria in comparison to the free enzyme. The obtained results demonstrated improved anti-biofilm efficacy and down regulation of tested biofilm genes for both SRP-loaded HA-Lys nanogel 100% and blank HA-Lys nanogel 100% compared to SRP 100%.