Polysaccharide Intercellular Adhesin Research Articles

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 of 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.

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.

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.

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.

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.

Drug efflux transporters in Staphylococcus pseudintermedius: in silico prediction and characterization of resistance.

To perform an in silico prediction of drug efflux pumps (EPs) in Staphylococcus pseudintermedius and investigate their role in conferring resistance to antibiotic and biocidal agents and biofilm formation. A S. pseudintermedius efflux mutant was obtained by stimulating an isogenic line (ATCC 49444) with increasing concentrations of an efflux system substrate. Changes in antimicrobial susceptibility and biofilm-forming capability were evaluated in the presence/absence of the EP inhibitors (EPIs) thioridazine and reserpine and the efflux activity was assayed by fluorometry. Homologues of EPs of Staphylococcus aureus and Staphylococcus epidermidis were searched by exploratory GenBank investigations. Gene expression analyses and sequencing were then conducted on selected genes. Susceptibility to chlorhexidine, gentamicin and ciprofloxacin, but not enrofloxacin, was affected by the increased efflux and it was variably restored by the EPIs. The efflux mutant showed much greater biofilm formation that the original strain, which was significantly inhibited by thioridazine and reserpine at MIC/2. A high expression of norA, which was mgrA-independent, was found in the S. pseudintermedius efflux mutant, apparently regulated by an 11 bp deletion in its promoter region, whilst lmrB was transitorily overexpressed. icaA, which encodes the polysaccharide intercellular adhesin forming the extracellular matrix of staphylococcal biofilm, was also up-regulated. EPs, particularly NorA, are supposed to have complex involvement in multiple stages of resistance development. Overexpression of EPs appears to be correlated with a remarkable increase of S. pseudintermedius biofilm production; however, the regulatory mechanisms remain to be explored.

Anti-biofilm effects of sinomenine against Staphylococcus aureus.

Staphylococcus aureus is a gram-positive foodborne pathogen capable of forming strong biofilms. This study identified that anti-biofilm natural compound against S. aureus. Sinomenine, a natural compound, showed significantly reduced biofilm formation (31.97-39.86%), but no effect on bacterial growth was observed. The dispersion of preformed biofilms was observed by confocal laser scanning microscopy (CLSM). qRT-PCR revealed that sinomenine treatment significantly up-regulated agrA by 3.8-fold and down-regulated icaA gene by 3.1-fold. These indicate that sinomenine treatment induces biofilm dispersal due to cell-cell adhesion, polysaccharide intercellular adhesin (PIA), and phenol-soluble modulin (PSM) peptides production. Our results suggest that sinomenine can be used as a promising agent for effectively controlling biofilm formation and dispersion, thereby making S. aureus more susceptible to the action of antimicrobial agents.

Lack of Direct Correlation between Biofilm Formation and Antimicrobial Resistance in Clinical Staphylococcus epidermidis Isolates from an Italian Hospital.

Staphylococcus epidermidis is an opportunistic pathogen and a frequent cause of nosocomial infections. In this work, we show that, among 51 S. epidermidis isolates from an Italian hospital, only a minority displayed biofilm formation, regardless of their isolation source (peripheral blood, catheter, or skin wounds); however, among the biofilm-producing isolates, those from catheters were the most efficient in biofilm formation. Interestingly, most isolates including strong biofilm producers displayed production levels of PIA (polysaccharide intercellular adhesin), the main S. epidermidis extracellular polysaccharide, similar to reference S. epidermidis strains classified as non-biofilm formers, and much lower than those classified as intermediate or high biofilm formers, possibly suggesting that high levels of PIA production do not confer a particular advantage for clinical isolates. Finally, while for the reference S. epidermidis strains the biofilm production clearly correlated with the decreased sensitivity to antibiotics, in particular, protein synthesis inhibitors, in our clinical isolates, such positive correlation was limited to tetracycline. In contrast, we observed an inverse correlation between biofilm formation and the minimal inhibitory concentrations for levofloxacin and teicoplanin. In addition, in growth conditions favoring PIA production, the biofilm-forming isolates showed increased sensitivity to daptomycin, clindamycin, and erythromycin, with increased tolerance to the trimethoprim/sulfamethoxazole association. The lack of direct correlation between the biofilm production and increased tolerance to antibiotics in S. epidermidis isolates from a clinical setting would suggest, at least for some antimicrobials, the possible existence of a trade-off between the production of biofilm determinants and antibiotic resistance.

Comparative profiling of agr locus, virulence, and biofilm-production genes of human and ovine non-aureus staphylococci

BackgroundIn a collaboration between animal and human health care professionals, we assessed the genetic characteristics shared by non-aureus staphylococci (NAS) infecting humans and dairy ewes to investigate their relatedness in a region concentrating half of the total National sheep stock. We examined by PCR 125 ovine and 70 human NAS for biofilm production, pyrogenic toxins, adhesins, autolysins genes, and accessory gene regulator (agr) locus. The microtiter plate assay (MPA) was used for the phenotypic screening of biofilm production. Ovine NAS included S. epidermidis, S. chromogenes, S. haemolyticus, S. simulans, S. caprae, S. warneri, S. saprophyticus, S. intermedius, and S. muscae. Human NAS included S. haemolyticus, S. epidermidis, S. hominis, S. lugdunensis, S. capitis, S. warneri, S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis.ResultsPhenotypically, 41 (32.8%) ovine and 24 (34.3%) human isolates were characterized as biofilm producers. Of the ovine isolates, 12 were classified as biofilm-producing while the remaining 29 as weak biofilm-producing. All 24 human isolates were considered weak biofilm-producing. Few S. epidermidis isolates harbored the icaA/D genes coding for the polysaccharide intercellular adhesin (PIA), while the bhp, aap, and embp genes coding biofilm accumulation proteins were present in both non-producing and biofilm-producing isolates. Fifty-nine sheep NAS (all S. epidermidis, 1 S. chromogenes, and 1 S. haemolyticus) and 27 human NAS (all S. epidermidis and 1 S. warneri) were positive for the agr locus: agr-3se (57.8%) followed by agr-1se (36.8%) predominated in sheep, while agr-1se (65.4%), followed by agr-2se (34.6%) predominated in humans.Concerning virulence genes, 40, 39.2, 47.2%, 52.8, 80 and 43.2% of the sheep isolates carried atlE, aae, sdrF, sdrG, eno and epbS respectively, against 37.1, 42.8, 32.8, 60, 100 and 100% of human isolates. Enterotoxins and tsst were not detected.ConclusionsConsiderable variation in biofilm formation ability was observed among NAS isolates from ovine and human samples. S. epidermidis was the best biofilm producer with the highest prevalence of adhesin-encoding genes.

Expression of biofilm-associated genes in Staphylococcus aureus during storage of platelet concentrates

Background and objectivesIn transfusion medicine, the safety of platelet concentrates (PCs) is a major concern on account of contamination, mostly with Staphylococcus species. One of the most common contaminants is Staphylococcus aureus, which forms bacterial biofilms in PCs, posing a safety risk for transfusion patients. In this study, we investigate the contributions to biofilm formation of eno, ebps, and fib genes encoding surface proteins and of genes from the ica operon (icaA and icaD) encoding polysaccharide intercellular adhesin (PIA), along with their expression in bacteria grown in glucose-supplemented trypticase soy broth (TSBg) and PCs. Materials and methodsTwo strains of S. aureus (2039 and 2110) captured during routine PC screening were tested for biofilm formation in TSBg and under PC storage conditions, with mRNA collected at five time points and analyzed to determine expression of eno, ebps, fib, icaA, and icaD and their contributions to biofilm formation in both media. ResultsIn TSBg, S. aureus strain 2039 formed weak biofilms while 2110 formed strong. biofilms; however, in PCs, both strains formed strong biofilms. During biofilm formation, expression levels of icaA and icaD in both strains were generally significantly higher in TSBg than PCs. In contrast, expression of eno, ebps, and fib genes tended to be significantly higher under PC storage conditions. ConclusionThis study demonstrated that expression of genes involved in biofilm formation can be affected by growth media. Further investigation is needed to understand biofilm formation in the PC milieu and enhance transfusion safety.

The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation.

Staphylococcus epidermidis is a common cause of device related infections on which pathogens form biofilms (i.e., multilayered cell populations embedded in an extracellular matrix). Here, we report that the transcription factor SpoVG is essential for the capacity of S. epidermidis to form such biofilms on artificial surfaces under in vitro conditions. Inactivation of spoVG in the polysaccharide intercellular adhesin (PIA) producing S. epidermidis strain 1457 yielded a mutant that, unlike its parental strain, failed to produce a clear biofilm in a microtiter plate-based static biofilm assay. A decreased biofilm formation capacity was also observed when 1457 ΔspoVG cells were co-cultured with polyurethane-based peripheral venous catheter fragments under dynamic conditions, while the cis-complemented 1457 ΔspoVG::spoVG derivative formed biofilms comparable to the levels seen with the wild-type. Transcriptional studies demonstrated that the deletion of spoVG significantly altered the expression of the intercellular adhesion (ica) locus by upregulating the transcription of the ica operon repressor icaR and down-regulating the transcription of icaADBC. Electrophoretic mobility shift assays (EMSA) revealed an interaction between SpoVG and the icaA-icaR intergenic region, suggesting SpoVG to promote biofilm formation of S. epidermidis by modulating ica expression. However, when mice were challenged with the 1457 ΔspoVG mutant in a foreign body infection model, only marginal differences in biomasses produced on the infected catheter fragments between the mutant and the parental strain were observed. These findings suggest that SpoVG is critical for the PIA-dependent biofilm formation of S. epidermis under in vitro conditions, but is largely dispensable for biofilm formation of this skin commensal under in vivo conditions.

Small-Molecule Compound SYG-180-2-2 to Effectively Prevent the Biofilm Formation of Methicillin-Resistant Staphylococcus aureus.

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) has augmented due to the abuse of antibiotics, bringing about difficulties in the treatment of infection especially with the formation of biofilm. Thus, it is essential to develop antimicrobials. Here we synthesized a novel small-molecule compound, which we termed SYG-180-2-2 (C21H16N2OSe), that had antibiofilm activity. The aim of this study was to demonstrate the antibiofilm effect of SYG-180-2-2 against clinical MRSA isolates at a subinhibitory concentration (4 μg/ml). In this study, it was showed that significant suppression in biofilm formation occurred with SYG-180-2-2 treatment, the inhibition ranged between 65.0 and 85.2%. Subsequently, confocal laser scanning microscopy and a bacterial biofilm metabolism activity assay further demonstrated that SYG-180-2-2 could suppress biofilm. Additionally, SYG-180-2-2 reduced bacterial adhesion and polysaccharide intercellular adhesin (PIA) production. It was found that the expression of icaA and other biofilm-related genes were downregulated as evaluated by RT-qPCR. At the same time, icaR and codY were upregulated when biofilms were treated with SYG-180-2-2. Based on the above results, we speculate that SYG-180-2-2 inhibits the formation of biofilm by affecting cell adhesion and the expression of genes related to PIA production. Above all, SYG-180-2-2 had no toxic effects on human normal alveolar epithelial cells BEAS-2B. Collectively, the small-molecule compound SYG-180-2-2 is a safe and effective antibacterial agent for inhibiting MRSA biofilm.