Intercellular Adhesin Research Articles

The roles of cell wall inhibition responsive protein CwrA in the pathogenicity of staphylococcus aureus

ABSTRACT The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of Staphylococcus aureus, causing a wide range of infectious diseases. Here, we characterized cwrA as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed cwrA deletion mutants in the genetic background of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Phenotypic analyses indicated that deletion of cwrA led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and β-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the ica operon activity in S. aureus. Furthermore, cwrA deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that cwrA deletion mutant more virulence than the parental strain because of its enhanced haemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and haemolytic activity in S. aureus.

Sub-inhibitory concentrations of tetrabromobisphenol A induce the biofilm formation of methicillin-resistant Staphylococcus aureus.

Biofilm formation by methicillin-resistant Staphylococcus aureus (MRSA) on indwelling medical devices complicates the treatment of infection. Tetrabromobisphenol A (TBBPA), a synthetic, lipophilic, halogenated aromatic compound widely used as an additive in plastics and electronic products, has raised environmental concerns due to its potential for bioaccumulation. This study investigated the impact of sub-inhibitory concentrations of TBBPA on MRSA biofilm formation. Crystal violet staining and confocal laser scanning microscopy analysis demonstrated that 1/8 MIC (0.5µg/mL) of TBBPA significantly stimulated MRSA biofilm formation (P < 0.0001). MTT assays indicated that the metabolic activity within the biofilms increased by 15.60-40.85% compared to untreated controls. Dot blot immunoassay, autolysis assay, and extracellular DNA (eDNA) quantification further revealed TBBPA enhanced the production of polysaccharide intercellular adhesin (PIA) and eDNA, which are key biofilm components. Additionally, TBBPA was found to enhance the production of staphyloxanthin, facilitating MRSA survival under oxidative conditions and in human whole blood. RT-qPCR analysis showed that TBBPA significantly upregulated genes associated with biofilm formation (icaA, atlA, sarA), staphyloxanthin biosynthesis (crtM and sigB), and oxidative stress responses (sodA and katA). These findings suggest that TBBPA promotes MRSA biofilm development and enhances bacterial resistance to adverse conditions, thereby potentially exacerbating risks to human health.

Diclofenac sodium effectively inhibits the biofilm formation of Staphylococcus epidermidis.

Staphylococcus epidermidis is an opportunistic pathogen commonly implicated in medical device-related infections. Its propensity to form biofilms not only leads to chronic infections but also exacerbates the issue of antibiotic resistance, necessitating high-dose antimicrobial treatments. In this study, we explored the use of diclofenac sodium, a non-steroidal anti-inflammatory drug, as an anti-biofilm agent against S. epidermidis. In this study, crystal violet staining and confocal laser scanning microscope analysis showed that diclofenac sodium, at subinhibitory concentration (0.4 mM), significantly inhibited biofilm formation in both methicillin-susceptible and methicillin-resistant S. epidermidis isolates. MTT assays demonstrated that 0.4 mM diclofenac sodium reduced the metabolic activity of biofilms by 25.21-49.01% compared to untreated controls. Additionally, the treatment of diclofenac sodium resulted in a significant decrease (56.01-65.67%) in initial bacterial adhesion, a crucial early phase of biofilm development. Notably, diclofenac sodium decreased the production of polysaccharide intercellular adhesin (PIA), a key component of the S. epidermidis biofilm matrix, in a dose-dependent manner. Real-time quantitative PCR analysis revealed that diclofenac sodium treatment downregulated biofilm-associated genes icaA, fnbA, and sigB and upregulated negative regulatory genes icaR and luxS, providing potential mechanistic insights. These findings indicate that diclofenac sodium inhibits S. epidermidis biofilm formation by affecting initial bacterial adhesion and the PIA synthesis. This underscores the potential of diclofenac sodium as a supplementary antimicrobial agent in combating staphylococcal biofilm-associated infections.

Polysaccharide intercellular adhesin and proper phospholipid composition are important for aggregation in Tetragenococcus halophilus SL10.

Aggregating strains of Tetragenococcus halophilus tend to be trapped during soy sauce mash-pressing process and are, therefore, critical for clear soy sauce production. However, the precise molecular mechanism involved in T. halophilus aggregation remains elusive. In previous studies, we isolated a number of aggregating strains, including T. halophilus AB4 and AL1, and showed that a cell surface proteinaceous aggregation factor is responsible for their aggregation phenotype. In the present study, we explored the role of polysaccharide intercellular adhesin (PIA) in aggregate formation in T. halophilus SL10, isolated from soy sauce. SL10 exhibited similar aggregation to AB4 and AL1 but formed a non-uniform precipitate with distinctive wrinkles at the bottom of the test tube, unlike AB4 and AL1. Insertion sequence mutations in each gene of the ica operon diminished aggregation and PIA production, highlighting the critical role of IcaADBC-mediated PIA production in T. halophilus aggregation. Furthermore, two non-aggregating cardiolipin synthase (cls) gene mutants with intact ica operon did not produce detectable PIA. Phospholipid composition analysis in cls mutants revealed a decrease in cardiolipin and an increase in phosphatidylglycerol levels, highlighting the association between phospholipid composition and PIA production. These findings provide evidence for the pivotal role of cls in PIA-mediated aggregation and lay the foundation for future studies to understand the intricate networks of the multiple aggregation factors governing microbial aggregation.IMPORTANCEAggregation, commonly observed in various microbes, triggers biofilm formation in pathogenic variants and plays a beneficial role in efficient food production in those used for food production. Here, we showed that Tetragenococcus halophilus, a microorganism used in soy sauce fermentation, forms aggregates in a polysaccharide intercellular adhesin (PIA)-mediated manner. Additionally, we unveiled the relationship between phospholipid composition and PIA production. This study provides evidence for the presence of aggregation factors in T. halophilus other than the proteinaceous aggregation factor and suggests that further understanding of the coordinated action of these factors may improve clarified soy sauce production.

The Functional Study of Response Regulator ArlR Mutants in Staphylococcus Aureus.

Staphylococcus aureus is a major cause of hospital-associated infections worldwide. The organism's ability to form biofilms has led to resistance against current treatment options such as beta-lactams, glycopeptides, and daptomycin. The ArlRS two-component system is a crucial regulatory system necessary for S. aureus autolysis, biofilm formation, capsule synthesis, and virulence. This study aims to investigate the role of the arlR deletion mutant in the detection and activation of S. aureus. We created an arlR deleted mutant and complementary strains and characterized their impact on the strains using partial growth measurement. The quantitative real-time PCR was performed to determine the expression of icaA, and the microscopic images of adherent cells were captured at the optical density of 600 to determine the primary bacterial adhesion. The biofilm formation assay was utilized to investigate the number of adherent cells using crystal violet staining. Eventually, the Triton X-100 autolysis assay was used to determine the influence of arlR on the cell autolytic activities. Our findings indicate that the deletion of arlR reduced the transcriptional expression of icaA but not icaR in the ica operon, leading to decrease in polysaccharide intercellular adhesin (PIA) synthesis. Compared to the wild-type and the complementary mutants, the arlR mutant exhibited decreased in biofilm production but increased autolysis. It concluded that the S. aureus response regulatory ArlR influences biofilm formation, agglutination, and autolysis. This work has significantly expanded our knowledge of the ArlRS two-component regulatory system and could aid in the development of novel antimicrobial strategies against S. aureus.

Ultrasonic-assisted extraction, anti-biofilm activity, and mechanism of action of Ku Shen (Sophorae Flavescentis Radix) extracts against Vibrio parahaemolyticus.

The objective of this study is to optimize the ultrasonic-assisted extraction process of Ku Shen (Sophorae Flavescentis Radix) extracts (KSE) against Vibrio parahaemolyticus and explore their anti-biofilm activity and mechanism of action. The ultrasonic-assisted extraction process of KSE optimized by single factor experiment, Box-Behnken design and response surface methodology was as follows: 93% ethanol as solvent, liquid/material ratio of 30 mL/g, ultrasonic power of 500 W, extraction temperature of 80°C and time of 30 min. Under these conditions, the diameter of inhibition circle of KSE was 15.60 ± 0.17 mm, which had no significant difference with the predicted value. The yield of dried KSE is 32.32 ± 0.57% and the content of total flavonoids in KSE was 57.02 ± 5.54%. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of KSE against V. parahaemolyticus were 0.25 and 0.5 mg/mL, respectively. Crystal violet staining, Congo red plate, spectrophotometry, CCK-8 and scanning electron microscopy were used to investigate the activity and mechanism of action of KSE against V. parahaemolyticus biofilm. The results showed that the sub-MIC of KSE could significantly inhibit biofilm formation, reduce the synthesis of polysaccharide intercellular adhesin (PIA) and the secretion of extracellular DNA. In addition, the inhibition rate of biofilm formation and clearance rate of mature biofilm of 1.0 mg/mL KSE were 85.32 and 74.04%, and the reduction rate of metabolic activity of developing and mature biofilm were 77.98 and 74.46%, respectively. These results were confirmed by visual images obtained by scanning electron microscopy. Therefore, KSE has the potential to further isolate the anti-biofilm agent and evaluate it for the preservation process of aquatic products.

Various mutations in icaR, the repressor of the icaADBC locus, occur in mucoid Staphylococcus aureus isolates recovered from the airways of people with cystic fibrosis

Staphylococcus aureus is one of the major pathogens isolated from the airways of people with cystic fibrosis (pwCF). Recently, we described a mucoid S. aureus phenotype from respiratory specimens of pwCF, which constitutively overproduced biofilm that consisted of polysaccharide intercellular adhesin (PIA) due to a 5bp-deletion (5bp-del) in the intergenic region of the intercellular adhesin (ica) locus.Since we were not able to identify the 5bp-del in mucoid isolates of two pwCF with long-term S. aureus persistence and in a number of mucoid isolates of pwCF from a prospective multicenter study, these strains were (i) characterized phenotypically, (ii) investigated for biofilm formation, and (iii) molecular typed by spa-sequence typing. To screen for mutations responsible for mucoidy, the ica operon of all mucoid isolates was analyzed by Sanger sequencing. Whole genome sequencing was performed for selected isolates.For all mucoid isolates without the 5 bp-del, various mutations in icaR, which is the transcriptional repressor of the icaADBC operon. Mucoid and non-mucoid strains belonged to the same spa-type. Transformation of PIA-overproducing S. aureus with a vector expressing the intact icaR gene restored the non-mucoid phenotype.Altogether, we demonstrated a new mechanism for the emergence of mucoid S. aureus isolates of pwCF.

Novel small-molecule compound YH7 inhibits the biofilm formation of Staphylococcus aureus in a sarX-dependent manner.

The emergence of antibiotic-resistant and biofilm-producing Staphylococcus aureus isolates presents major challenges for treating staphylococcal infections. Biofilm inhibition is an important anti-virulence strategy. In this study, a novel maleimide-diselenide hybrid compound (YH7) was synthesized and demonstrated remarkable antimicrobial activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in both planktonic cultures and biofilms. The minimum inhibitory concentration (MIC) of YH7 for S. aureus isolates was 16 µg/mL. Quantification of biofilms demonstrated that the sub-MIC (4 µg/mL) of YH7 significantly inhibits biofilm formation in both MSSA and MRSA. Confocal laser scanning microscopy analysis further confirmed the biofilm inhibitory potential of YH7. YH7 also significantly suppressed bacterial adherence to A549 cells. Moreover, YH7 treatment significantly inhibited S. aureus colonization in nasal tissue of mice. Preliminary mechanistic studies revealed that YH7 exerted potent biofilm-suppressing effects by inhibiting polysaccharide intercellular adhesin (PIA) synthesis, rather than suppressing bacterial autolysis. Real-time quantitative PCR data indicated that YH7 downregulated biofilm formation-related genes (clfA, fnbA, icaA, and icaD) and the global regulatory gene sarX, which promotes PIA synthesis. The sarX-dependent antibiofilm potential of YH7 was validated by constructing S. aureus NCTC8325 sarX knockout and complementation strains. Importantly, YH7 demonstrated a low potential to induce drug resistance in S. aureus and exhibited non-toxic to rabbit erythrocytes, A549, and BEAS-2B cells at antibacterial concentrations. In vivo toxicity assays conducted on Galleria mellonella further confirmed that YH7 is biocompatible. Overall, YH7 demonstrated potent antibiofilm activity supports its potential as an antimicrobial agent against S. aureus biofilm-related infections. IMPORTANCE Biofilm-associated infections, characterized by antibiotic resistance and persistence, present a formidable challenge in healthcare. Traditional antibacterial agents prove inadequate against biofilms. In this study, the novel compound YH7 demonstrates potent antibiofilm properties by impeding the adhesion and the polysaccharide intercellular adhesin production of Staphylococcus aureus. Notably, its exceptional efficacy against both methicillin-resistant and methicillin-susceptible strains highlights its broad applicability. This study highlights the potential of YH7 as a novel therapeutic agent to address the pressing issue of biofilm-driven infections.

Functional Study of desKR: a Lineage-Specific Two-Component System Positively Regulating Staphylococcus aureus Biofilm Formation.

Biofilms significantly contribute to the persistence and antibiotic resistance of Staphylococcus aureus infections. However, the regulatory mechanisms governing biofilm formation of S. aureus remain not fully elucidated. This study aimed to investigate the function of the S. aureus lineage-specific two-component system, desKR, in biofilm regulation and pathogenicity. Bioinformatic analysis was conducted to assess the prevalence of desKR across various S. aureus lineages and to examine its structural features. The impact of desKR on S. aureus pathogenicity was evaluated using in vivo mouse models, including skin abscess, bloodstream infection, and nasal colonization models. Crystal violet staining and confocal laser scanning microscopy were utilized to examine the impact of desKR on S. aureus biofilm formation. Mechanistic insights into desKR-mediated biofilm regulation were investigated by quantifying polysaccharide intercellular adhesin (PIA) production, extracellular DNA (eDNA) release, autolysis assays, and RT-qPCR. The prevalence of desKR varied among different S. aureus lineages, with notably low carriage rates in ST398 and ST59 lineages. Deletion of desKR in NCTC8325 strain resulted in decreased susceptibility to β-lactam and glycopeptide antibiotics. Although desKR did not significantly affect acute pathogenicity, the ΔdesKR mutant exhibited significantly reduced nasal colonization and biofilm-forming ability. Overexpression of desKR in naturally desKR-lacking strains (ST398 and ST59) enhanced biofilm formation, suggesting a lineage-independent effect. Phenotypic assays further revealed that the ΔdesKR mutant showed reduced PIA production, decreased eDNA release, and lower autolysis rates. RT-qPCR indicated significant downregulation of icaA, icaD, icaB, and icaC genes, along with upregulation of icaR, whereas autolysis-related genes remained unchanged. The desKR two-component system positively regulates S. aureus biofilm formation in a lineage-independent manner, primarily by modulating PIA synthesis via the ica operon. These findings provide new insights into the molecular mechanisms of biofilm formation in S. aureus and highlight desKR as a potential target for therapeutic strategies aimed at combating biofilm-associated infections.

Ciprofloxacin enhances the biofilm formation of Staphylococcus aureus via an agrC-dependent mechanism.

Staphylococcus aureus readily forms biofilms on host tissues and medical devices, enabling its persistence in chronic infections and resistance to antibiotic therapy. The accessory gene regulator (Agr) quorum sensing system plays a key role in regulating S. aureus biofilm formation. This study reveals the widely used fluoroquinolone antibiotic, ciprofloxacin, strongly stimulates biofilm formation in methicillin-resistant S. aureus, methicillin-sensitive S. aureus, and clinical isolates with diverse genetic backgrounds. Crystal violet staining indicated that ciprofloxacin induced a remarkable 12.46- to 15.19-fold increase in biofilm biomass. Confocal laser scanning microscopy revealed that ciprofloxacin induced denser biofilms. Phenotypic assays suggest that ciprofloxacin may enhance polysaccharide intercellular adhesin production, inhibit autolysis, and reduce proteolysis during the biofilm development, thus promoting initial adhesion and enhancing biofilm stability. Mechanistically, ciprofloxacin significantly alters the expression of various biofilm-related genes (icaA, icaD, fnbA, fnbB, eap, emp) and regulators (agrA, saeR). Gene knockout experiments revealed that deletion of agrC, rather than saeRS, abolishes the ciprofloxacin-induced enhancement of biofilm formation, underscoring the key role of agrC. Thermal shift assays showed ciprofloxacin binds purified AgrC protein, thereby inhibiting the Agr system. Molecular docking results further support the potential interaction between ciprofloxacin and AgrC. In summary, subinhibitory concentrations of ciprofloxacin stimulate S. aureus biofilm formation via an agrC-dependent pathway. This inductive effect may facilitate local infection establishment and bacterial persistence, ultimately leading to therapeutic failure.

Characterization of biofilm formation in uropathogenic Staphylococcus aureus and their association with antibiotic resistance

IntroductionThe increase in antibiotic resistance and biofilm formation in Staphylococcus aureus is a serious public health problem, complicating the treatment of infections caused by these bacteria, including urinary tract infections (UTIs). This research aimed to assess the correlation between antibiotic resistance and the capacity for biofilm formation in S. aureus strains isolated from patients with UTIs. MethodsThe antibiotic susceptibility testing was conducted using disc diffusion and broth micro-dilution method. The blaZ and mecA genes were investigated in all penicillin and cefoxitin-resistant S. aureus strains, using PCR. Biofilm formation was investigated using the Congo red agar and microtitre plate methods. All S. aureus strains were screened for genes encoding adhesion and biofilm formation (spa, fnb AB, clfA, bap) and intercellular adhesin (icaADB and icaR). ResultsAmong 20 strains of S. aureus, 55% were found to be multidrug-resistant (MDR) and methicillin-resistant (MRSA). All strains were biofilm producers, including 55% moderate biofilm producers and 35% strong biofilm producers. A significant correlation (p-value <0.01) was observed between MDR and non-MDR strains in terms of biofilm production. All strains were sensitive to linezolid, and 20% exhibited the constitutive clindamycin resistance phenotype. The spa, fnbB, and clfA genes were present in all strains followed by the bap (95%) and fnbA (90%). The icaA (90%) and icaB (60%) genes were most detected. The coexistence of icaA, icaADB, and icaAD genes was significantly associated with MRSA strains. In contrast, the icaR gene was significantly detectable in the methicillin-susceptible group of strains. ConclusionThis study uncovered a significant association between the formation of biofilms by uropathogenic S. aureus and antibiotic resistance, highlighting a noteworthy concern for public health.

Increased Activity of β-Lactam Antibiotics in Combination with Carvacrol against MRSA Bacteremia and Catheter-Associated Biofilm Infections.

β-Lactam antibiotics are the mainstay for the treatment of staphylococcal infections, but their utility is greatly limited by the emergence and rapid dissemination of methicillin-resistant Staphylococcus aureus (MRSA). Herein, we evaluated the ability of the plant-derived monoterpene carvacrol to act as an antibiotic adjuvant, revitalizing the anti-MRSA activity of β-lactam antibiotics. Increased susceptibility of MRSA to β-lactam antibiotics and significant synergistic activities were observed with carvacrol-based combinations. Carvacrol significantly inhibited MRSA biofilms and reduced the production of exopolysaccharide, polysaccharide intercellular adhesin, and extracellular DNA and showed synergistic biofilm inhibition in combination with β-lactams. Transcriptome analysis revealed profound downregulation in the expression of genes involved in two-component systems and S. aureus infection. Mechanistic studies indicate that carvacrol inhibits the expression of staphylococcal accessory regulator sarA and interferes with SarA-mecA promoter binding that decreases mecA-mediated β-lactam resistance. Consistently, the in vivo experiment also supported that carvacrol restored MRSA sensitivity to β-lactam antibiotic treatments in both murine models of bacteremia and biofilm-associated infection. Our results indicated that carvacrol has a potential role as a combinatorial partner with β-lactam antibiotics to address MRSA infections.

A novel small-molecule compound S-342-3 effectively inhibits the biofilm formation of Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen in both community and hospital settings that often causes persistent and recurrent infections. The continuous emergence of multidrug-resistant strains and the lag in antibiotic development make the treatment of S. aureus more challenging. The biofilm formation of S. aureus is an important reason for persistent infection. In this study, we demonstrated that a small-molecule compound S-342-3 can effectively inhibit the biofilm formation of S. aureus. The crystal violet semi-quantitative assays revealed that at a sub-minimum inhibitory concentration of 4 µg/mL, S-342-3 reduced S. aureus biofilm mass by 57.43%, 52.14%, and 25.49%. Using confocal laser scanning microscopy, we observed that the biofilm was more incompact and less uniform upon the treatment of S-342-3. At a sub-inhibitory concentration (4 µg/mL), the S-342-3 can effectively reduce the production of polysaccharide intercellular adhesin (PIA) and suppress the initial adhesion of cells in the biofilm. Consistently, the results of RT-qPCR revealed that the expression of genes associated with biofilm formation was downregulated by S-342-3 (P < 0.05). However, we found that treatment with S-342-3 resulted in a significant decrease in the expression of global regulatory genes agrA and sarA (P < 0.05), which play a key role in promoting cell surface attachment and PIA production in S. aureus biofilms. Also importantly, we experimentally proved that the S-342-3 was not toxic to A549 alveolar epithelial cells and the Galleria mellonella larvae. Collectively, these results suggest that the S-342-3 may be a promising anti-biofilm drug candidate with excellent biosafety, which can be further explored for its practical application in health care. IMPORTANCE Biofilms are an important virulence factor in Staphylococcus aureus and are characterized by a structured microbial community consisting of bacterial cells and a secreted extracellular polymeric matrix. Inhibition of biofilm formation is an effective measure to control S. aureus infection. Here, we have synthesized a small molecule compound S-342-3, which exhibits potent inhibition of biofilm formation in both MRSA and MSSA. Further investigations revealed that S-342-3 exerts inhibitory effects on biofilm formation by reducing the production of polysaccharide intercellular adhesin and preventing bacterial adhesion. Our study has confirmed that the inhibitory effect of S-342-3 on biofilm is achieved by downregulating the expression of genes responsible for biofilm formation. In addition, S-342-3 is non-toxic to Galleria mellonella larvae and A549 cells. Consequently, this study demonstrates the efficacy of a biologically safe compound S-342-3 in inhibiting biofilm formation in S. aureus, thereby providing a promising antibiofilm agent for further research.

Regulation of σB-Dependent Biofilm Formation in Staphylococcus aureus through Strain-Specific Signaling Induced by Diosgenin.

Staphylococcus aureus is a commensal skin bacterium and a causative agent of infectious diseases. Biofilm formation in S. aureus is a mechanism that facilitates the emergence of resistant strains. This study proposes a mechanism for the regulation of biofilm formation in S. aureus through strain-specific physiological changes induced by the plant steroid diosgenin. A comparison of diosgenin-induced changes in the expression of regulatory genes associated with physiological changes revealed the intracellular regulatory mechanisms involved in biofilm formation. Diosgenin reduced biofilm formation in S. aureus ATCC 6538 and methicillin-resistant S. aureus (MRSA) CCARM 3090 by 39% and 61%, respectively. Conversely, it increased biofilm formation in S. aureus ATCC 29213 and MRSA CCARM 3820 by 186% and 582%, respectively. Cell surface hydrophobicity and extracellular protein and carbohydrate contents changed in a strain-specific manner in response to biofilm formation. An assessment of the changes in gene expression associated with biofilm formation revealed that diosgenin treatment decreased the expression of icaA and spa and increased the expression of RNAIII, agrA, sarA, and sigB in S. aureus ATCC 6538 and MRSA CCARM 3090; however, contrasting gene expression changes were noted in S. aureus ATCC 29213 and MRSA CCARM 3820. These results suggest that a regulatory mechanism of biofilm formation is that activated sigB expression sequentially increases the expression of sarA, agrA, and RNAIII. This increased RNAIII expression decreases the expression of spa, a surface-associated adhesion factor. An additional regulatory mechanism of biofilm formation is that activated sigB expression decreases the expression of an unknown regulator that increases the expression of icaA. This in turn decreases the expression of icaA, which decreases the synthesis of polysaccharide intercellular adhesins and ultimately inhibits biofilm formation. By assessing strain-specific contrasting regulatory signals induced by diosgenin in S. aureus without gene mutation, this study elucidated the signal transduction mechanisms that regulate biofilm formation based on physiological and gene expression changes.

Sponge-derived fatty acids inhibit biofilm formation of MRSA and MSSA by down-regulating biofilm-related genes specific to each pathogen.

A promising approach for the development of next-generation antimicrobials is to shift their target from causing bacterial death to inhibiting virulence. Marine sponges are an excellent potential source of bioactive anti-virulence molecules (AVM). We screened fractions prepared from 26 samples of Irish coastal sponges for anti-biofilm activity against clinically relevant pathogens. Fifteen fractions from eight sponge species inhibited biofilm of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and/or Listeria monocytogenes without causing growth inhibition. Gas chromatograph/mass spectroscopy analyses of Mycale contarenii fractions revealed the presence of myristic acid and oleic acid. These fatty acids repressed transcription of the fibronectin-binding protein fnbA and fnbB genes and the polysaccharide intercellular adhesin icaADBC operon, which are required for MRSA and MSSA biofilm formation, respectively. This study illustrates the potential of AVM from Irish coastal sponges to specifically target bacterial virulence phenotypes, in this case, repression of biofilm formation via decreased transcription of biofilm-associated genes in MSSA and MRSA.

Targeting and arginine-driven synergizing photodynamic therapy with nutritional immunotherapy nanosystems for combating MRSA biofilms.

The resistance and immune escape of methicillin-resistant Staphylococcus aureus (MRSA) biofilms cause recalcitrant infections. Here, we design a targeting and synergizing cascade PDT with nutritional immunotherapy nanosystems (Arg-PCN@Gel) containing PCN-224 as PDT platform for providing reactive oxygen species (ROS), incorporating arginine (Arg) as nitric oxide (NO) donor to cascade with ROS to produce more lethal ONOO- and promote immune response, and coating with gelatin as targeting agent and persistent Arg provider. The nanosystems adhered to the autolysin of MRSA and inhibited Arg metabolism by down-regulating icdA and icaA. It suppressed polysaccharide intercellular adhesin and extracellular DNA synthesis to prevent biofilm formation. The NO broke mature biofilms and helped ROS and ONOO- penetrate into biofilms to inactivate internal MRSA. Arg-PCN@Gel drove Arg to enhance immunity via inducible NO synthase/NO axis and arginase/polyamine axis and achieve efficient target treatment in MRSA biofilm infections. The targeting and cascading PDT synergized with nutritional immunotherapy provide an effective promising strategy for biofilm-associated infections.

Mupirocin enhances the biofilm formation of Staphylococcus epidermidis in an atlE-dependent manner

The pathogenicity of Staphylococcus epidermidis is largely attributed to its exceptional ability to form biofilms. Here, we report that mupirocin, an antimicrobial agent widely used for staphylococcal decolonization and anti-infection, strongly stimulates the biofilm formation of S. epidermidis. Although the polysaccharide intercellular adhesin (PIA) production was unaffected, mupirocin significantly facilitated extracellular DNA (eDNA) release by accelerating autolysis, thereby positively triggering cell surface attachment and intercellular agglomeration during biofilm development. Mechanistically, mupirocin regulated the expression of genes encoding for the autolysin AtlE as well as the programmed cell death system CidA-LrgAB. Critically, through gene knockout, we found out that deletion of atlE, but not cidA or lrgA, abolished the enhancement of biofilm formation and eDNA release in response to mupirocin treatment, indicating that atlE is required for this effect. In Triton X-100 induced autolysis assay, mupirocin treated atlE mutant displayed a slower autolysis rate compared with the wild-type strain and complementary strain. Therefore, we concluded that subinhibitory concentrations of mupirocin enhance the biofilm formation of S. epidermidis in an atlE dependent manner. This induction effect could conceivably be responsible for some of the more unfavorable outcomes of infectious diseases.

A genetic regulatory see-saw of biofilm and virulence in MRSA pathogenesis.

Staphylococcus aureus is one of the most common opportunistic human pathogens causing several infectious diseases. Ever since the emergence of the first methicillin-resistant Staphylococcus aureus (MRSA) strain decades back, the organism has been a major cause of hospital-acquired infections (HA-MRSA). The spread of this pathogen across the community led to the emergence of a more virulent subtype of the strain, i.e., Community acquired Methicillin resistant Staphylococcus aureus (CA-MRSA). Hence, WHO has declared Staphylococcus aureus as a high-priority pathogen. MRSA pathogenesis is remarkable because of the ability of this "superbug" to form robust biofilm both in vivo and in vitro by the formation of polysaccharide intercellular adhesin (PIA), extracellular DNA (eDNA), wall teichoic acids (WTAs), and capsule (CP), which are major components that impart stability to a biofilm. On the other hand, secretion of a diverse array of virulence factors such as hemolysins, leukotoxins, enterotoxins, and Protein A regulated by agr and sae two-component systems (TCS) aids in combating host immune response. The up- and downregulation of adhesion genes involved in biofilm formation and genes responsible for synthesizing virulence factors during different stages of infection act as a genetic regulatory see-saw in the pathogenesis of MRSA. This review provides insight into the evolution and pathogenesis of MRSA infections with a focus on genetic regulation of biofilm formation and virulence factors secretion.

Optimized Extraction, Identification and Anti-Biofilm Action of Wu Wei Zi (Fructus Schisandrae Chinensis) Extracts against Vibrio parahaemolyticus

The pathogenicity of foodborne Vibrio parahaemolyticus is a major concern for global public health. This study aimed to optimize the liquid-solid extraction of Wu Wei Zi extracts (WWZE) against Vibrio parahaemolyticus, identify its main components, and investigate the anti-biofilm action. The extraction conditions optimized by the single-factor test and response surface methodology were ethanol concentration of 69%, temperature at 91 °C, time of 143 min, and liquid-solid ratio of 20:1 mL/g. After high performance liquid chromatography (HPLC) analysis, it was found that the main active ingredients of WWZE were schisandrol A, schisandrol B, schisantherin A, schisanhenol, and schisandrin A-C. The minimum inhibitory concentration (MIC) of WWZE, schisantherin A, and schisandrol B measured by broth microdilution assay was 1.25, 0.625, and 1.25 mg/mL, respectively, while the MIC of the other five compounds was higher than 2.5 mg/mL, indicating that schisantherin A and schizandrol B were the main antibacterial components of WWZE. Crystal violet, Coomassie brilliant blue, Congo red plate, spectrophotometry, and Cell Counting Kit-8 (CCK-8) assays were used to evaluate the effect of WWZE on the biofilm of V. parahaemolyticus. The results showed that WWZE could exert its dose-dependent potential to effectively inhibit the formation of V. parahaemolyticus biofilm and clear mature biofilm by significantly destroying the cell membrane integrity of V. parahaemolyticus, inhibiting the synthesis of intercellular polysaccharide adhesin (PIA), extracellular DNA secretion, and reducing the metabolic activity of biofilm. This study reported for the first time the favorable anti-biofilm effect of WWZE against V. parahaemolyticus, which provides a basis for deepening the application of WWZE in the preservation of aquatic products.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.