Polysaccharide Intercellular Adhesion Research Articles

In silico design and mechanistic study of niosome-encapsulated curcumin against multidrug-resistant Staphylococcus aureus biofilms

Curcumin, an important natural component of turmeric, has been known for a long time for its antimicrobial properties. This study aimed to investigate the anti-biofilm action of the niosome-encapsulated curcumin and explore the involved anti-biofilm mechanism. In silico investigations of ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) were first performed to predict the suitability of curcumin for pharmaceutical application. Curcumin showed low toxicity but at the same time, low solubility and low stability, which, in turn, might reduce its antimicrobial activity. To overcome these intrinsic limitations, curcumin was encapsulated using a biocompatible niosome system, and an encapsulation efficiency of 97% was achieved. The synthesized curcumin-containing niosomes had a spherical morphology with an average diameter of 178 nm. The niosomal curcumin was capable of reducing multi-drug resistant (MDR) Staphylococcus aureus biofilm 2–4-fold compared with the free curcumin. The encapsulated curcumin also demonstrated no significant cytotoxicity on the human foreskin fibroblasts. To understand the interaction between curcumin and S. aureus biofilm, several biofilm-related genes were analyzed for their expression. N-acetylglucosaminyl transferase (IcaD), a protein involved in the production of polysaccharide intercellular adhesion and known to play a function in biofilm development, was found to be downregulated by niosomal curcumin and showed high binding affinity (-8.3 kcal/mol) with curcumin based on molecular docking analysis. Our study suggests that the niosome-encapsulated curcumin is a promising approach for the treatment of MDR S. aureus biofilm and can be extended to biofilms caused by other pathogens.

Staphylococcus epidermidis biofilms undergo metabolic and matrix remodeling under nitrosative stress.

Staphylococcus epidermidis is a commensal skin bacterium that forms host- and antibiotic-resistant biofilms that are a major cause of implant-associated infections. Most research has focused on studying the responses to host-imposed stresses on planktonic bacteria. In this work, we addressed the open question of how S. epidermidis thrives on toxic concentrations of nitric oxide (NO) produced by host innate immune cells during biofilm assembly. We analyzed alterations of gene expression, metabolism, and matrix structure of biofilms of two clinical isolates of S. epidermidis, namely, 1457 and RP62A, formed under NO stress conditions. In both strains, NO lowers the amount of biofilm mass and causes increased production of lactate and decreased acetate excretion from biofilm glucose metabolism. Transcriptional analysis revealed that NO induces icaA, which is directly involved in polysaccharide intercellular adhesion (PIA) production, and genes encoding proteins of the amino sugar pathway (glmM and glmU) that link glycolysis to PIA synthesis. However, the strains seem to have distinct regulatory mechanisms to boost lactate production, as NO causes a substantial upregulation of ldh gene in strain RP62A but not in strain 1457. The analysis of the matrix components of the staphylococcal biofilms, assessed by confocal laser scanning microscopy (CLSM), showed that NO stimulates PIA and protein production and interferes with biofilm structure in a strain-dependent manner, but independently of the Ldh level. Thus, NO resistance is attained by remodeling the staphylococcal matrix architecture and adaptation of main metabolic processes, likely providing in vivo fitness of S. epidermidis biofilms contacting NO-proficient macrophages.

Metabolomic profiling of bacterial biofilm: trends, challenges, and an emerging antibiofilm target.

Biofilm-related infections substantially contribute to bacterial illnesses, with estimates indicating that at least 80% of such diseases are linked to biofilms. Biofilms exhibit unique metabolic patterns that set them apart from their planktonic counterparts, resulting in significant metabolic reprogramming during biofilm formation. Differential glycolytic enzymes suggest that central metabolic processes are markedly different in biofilms and planktonic cells. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is highly expressed in Staphylococcus aureus biofilm progenitors, indicating that changes in glycolysis activity play a role in biofilm development. Notably, an important consideration is a correlation between elevated cyclic di-guanylate monophosphate (c-di-GMP) activity and biofilm formation in various bacteria. C-di-GMP plays a critical role in maintaining the persistence of Pseudomonas aeruginosa biofilms by regulating alginate production, a significant biofilm matrix component. Furthermore, it has been demonstrated that S. aureus biofilm development is initiated by several tricarboxylic acid (TCA) intermediates in a FnbA-dependent manner. Finally, Glucose 6-phosphatase (G6P) boosts the phosphorylation of histidine-containing protein (HPr) by increasing the activity of HPr kinase, enhancing its interaction with CcpA, and resulting in biofilm development through polysaccharide intercellular adhesion (PIA) accumulation and icaADBC transcription. Therefore, studying the metabolic changes associated with biofilm development is crucial for understanding the complex mechanisms involved in biofilm formation and identifying potential targets for intervention. Accordingly, this review aims to provide a comprehensive overview of recent advances in metabolomic profiling of biofilms, including emerging trends, prevailing challenges, and the identification of potential targets for anti-biofilm strategies.

To study drug resistance & biofilm production in gram negative isolates from clinical samples

Biofilms are groups of microorganism encased in a matrix of extracellular polysaccharide (slime), called polysaccharide intercellular adhesion (PIA). Bacteria commonly involved include , , , and . The present study was performed to identify antibiotic resistance pattern & their ability to form biofilm in gram negative clinical isolates. All clinical samples received in laboratory for microbial culture during study period of 12 months (2017 to 2018) were included in this study Antibiotic susceptibility testing, ESBL & MBL detection was done for clinical isolates. Biofilm productions were determined by Congo red agar method, Christenson’s Test Tube method and Tissue culture plate method. 327 gram negative isolates were detected. Maximum were (32.72%) followed by (28.44%), (16.51%), (16.51%), Citrobacter species (3.97%). Maximum isolates showed resistance to ampicillin (93.27%) followed by amoxiclave (87.46%), ceftazidime (74%). Out of 327 GNB isolates, biofilm produced by 64 (19.57%) isolates by Tissue culture plate (TCP) method, 38(11.62%) by Congo red agar (CRA) method and 23 (7.03%) by Tube method. Maximum biofilm were detected in (24.29%). There is increase prevalence of multidrug resistant& biofilm forming bacteria. The routine monitoring of multidrug resistance pattern & biofilm detection can be recommended in clinical laboratories to guide proper antibiotic treatment.

Development of an artificial synovial fluid useful for studying Staphylococcus epidermidis joint infections.

Staphylococcus epidermidis is a major causative agent of prosthetic joint infections (PJI). The ability to form biofilms supports this highly selective pathogenic potential. In vitro studies essentially relying on phenotypic assays and genetic approaches have provided a detailed picture of the molecular events contributing to biofilm assembly. A major limitation in these studies is the use of synthetic growth media, which significantly differs from the environmental conditions S. epidermidis encounters during host invasion. Building on evidence showing that growth in serum substantially affects S. epidermidis gene expression profiles and phenotypes, the major aim of this study was to develop and characterize a growth medium mimicking synovial fluid, thereby facilitating research addressing specific aspects related to PJI. Using fresh human plasma, a protocol was established allowing for the large-scale production of a medium that by biochemical analysis matches key characteristics of synovial fluid and therefore is referred to as artificial synovial fluid (ASF). By analysis of biofilm-positive, polysaccharide intercellular adhesion (PIA)-producing S. epidermidis 1457 and its isogenic, PIA- and biofilm-negative mutant 1457-M10, evidence is provided that the presence of ASF induces cluster formation in S. epidermidis 1457 and mutant 1457-M10. Consistent with the aggregative properties, both strains formed multilayered biofilms when analyzed by confocal laser scanning microscopy. In parallel to the phenotypic findings, expression analysis after growth in ASF found upregulation of genes encoding for intercellular adhesins (icaA, aap, and embp) as well as atlE, encoding for the major cell wall autolysin being responsible for eDNA release. In contrast, growth in ASF was associated with reduced expression of the master regulator agr. Collectively, these results indicate that ASF induces expression profiles that are able to support intercellular adhesion in both PIA-positive and PIA-negative S. epidermidis. Given the observation that ASF overall induced biofilm formation in a collection of S. epidermidis isolates from PJI, the results strongly support the idea of using growth media mimicking host environments. ASF may play an important role in future studies related to the pathogenesis of S. epidermidis PJI.

Four temporin-derived peptides exhibit antimicrobial and antibiofilm activities against methicillin-resistant.

Temporin-GHa (GHa) was cloned from , showing a weak antimicrobial activity. In order to improve its bactericidal efficacy, GHaR6R, GHaR7R, GHaR8R and GHaR9W were designed and synthesized. Compared to the parent peptide, the GHa-derived peptides show potent antimicrobial activities against methicillin-resistant (MRSA), which is the main pathogen with high morbidity and mortality that causes various infections in humans. These peptides exert bactericidal actions on MRSA by permeabilizing the cytoplasmic membranes and damaging membrane integrity. All of the four peptides exhibit excellent stability under harsh conditions, including extreme temperature and salts. Furthermore, they inhibit the formation of biofilm and eradicate mature biofilm of MRSA. The GHa-derived peptides decrease bacterial surface hydrophobicity, autoaggregation and polysaccharide intercellular adhesion synthesis in concentration-dependent manner. Real-time quantitative reverse transcription PCR analysis revealed that the peptides downregulate the expression of adhesion genes involved in biofilm formation. Except for GHaR7R, the other three peptides have low hemolytic toxicity against human erythrocytes. In the presence of human erythrocytes, GHaR7R, GHaR8R and GHaR9W interact with MRSA preferentially. GHaR6R, GHaR8R and GHaR9W show less toxicity toward normal cells HL-7702 and hFOB1.19. These results suggest that the GHa-derived peptides may be promising antimicrobial candidates against MRSA infections.

Staphylococcus aureus biofilm inhibition by high voltage prick electrostatic field (HVPEF) and the mechanism investigation

The study was to investigate the inhibitory effect and mechanism of high voltage prick electrostatic field (HVPEF) on Staphylococcus aureus biofilms. Results showed that HVPEF effectively inactivated 24-h and 48-h established S. aureus biofilms, and the effect was verified on different food-contact materials. Confocal laser scanning microscopy and scanning electron microscopy analysis suggested that HVPEF disintegrated the established biofilms by killing the embedded bacteria, but it hardly reduced the bacteria adhesion. HVPEF also effectively inhibit the formation of S. aureus biofilms, the effects varied with electric voltage, treatment time and biofilm culture conditions. The direct effect of HVPEF on planktonic S. aureus was a possible mode of biofilm formation inhibition. HVPEF also suppressed biofilm formation by reducing the release of key compositions of extracellular polymeric substance, including extracellular DNA (eDNA), protein and polysaccharide intercellular adhesion (PIA), and regulating the expression of biofilm formation related genes (icaA, ebh, cidA, sarA, icaR and sigB). We propose HVPEF as a novel method to inhibit bacteria biofilm, based on the results, HVPEF has positive effects to prevent biofilm-associated contamination of S. aureus.

Effect of Ultraviolet Light on the Expression of icaD Gene in Staphylococcus aureus Local Isolates in Iraq.

The current study was designed to reveal the impact of ultraviolet (UV) light on polysaccharide intercellular adhesion of the icaD gene expression and its relation to biofilm as virulence markers in Staphylococcus aureus. In total, 37 isolates of S. aureus were gathered from various bacterial infection sources, and 26 S. aureus isolates were definite to icaD gene using the Polymerase Chain Reaction (PCR) technique. Prior to and after exposure to UV light, RNAs from the isolates were extracted which had strong biofilm formation by crystal violet staining assay, and they were then exposed to quantitative real-time PCR (qPCR). In vitro, the survival of isolates was evaluated after the exposition to several periods of the UV light power via counting the number of colonies. The results showed that the exposure to the UV light at 320 nm for 2, 4, 6, 8, and 10 min had inhibitory effects on the bacterial growth. The sub-lethal exposure time was 8 min. The rapid reduction of bacterial survival rates was associated with the increment irradiation time of UV power from 5 to 1.24 log10 CFU/ml. In general, down regulation of the icaD gene expression was decreased upon exposure to the UV light which was used as a physical agent for controlling the bacterial infection and biofilm formation.

Phenotypic PIA-Dependent Biofilm Production by Clinical Non-Typeable Staphylococcus aureus Is Not Associated with the Intensity of Inflammation in Mammary Gland: A Pilot Study Using Mouse Mastitis Model.

Simple SummaryStaphylococcus aureus-associated human clinical infections are predominantly caused by the encapsulated strains, with non-typeable strains representing less than 25%. In contrast, 80% of the S. aureus from bovine mastitis cases are non-typeable as they do not possess the Capsular Types 1, 2, 5, and 8. In our previous studies, it was demonstrated that the extent of mammary tissue damage was associated with the strength of biofilms formed by encapsulated S. aureus strains. This study assesses the impact of biofilm formation, as a virulence factor of non-typeable Staphylococcus aureus, causing mammary tissue damage in a mouse mastitis model. The study demonstrates no association between the strength of biofilm production by non-typeable S. aureus and the mammary tissue damage. However, the mice infected with strong biofilm producing non-typeable S. aureus died 6h earlier than those infected with weak biofilm producing non-typeable S. aureus suggesting the role of biofilm in the advancement of the time of mice mortality.Non-typeable (NT) Staphylococcus aureus strains are associated with chronic bovine mastitis. This study investigates the impact of biofilm formation by clinical NT S. aureus on cytokine production and mammary tissue damage by using a mouse mastitis model. Mice infected with two different NT S. aureus strains with strong and weak biofilm forming potential demonstrated identical clinical symptoms (moderate), minimal inflammatory infiltrates, and tissue damage (level 1 histopathological changes) in the mammary glands. However, the S. aureus load in the mammary glands of mice and the level of pro-inflammatory cytokines (IL-1β, IL-6, IL-12, IL-17 and IFN-γ) in serum were significantly higher (p ≤ 0.05) in those infected with the strong biofilm forming NT S. aureus strain. The level of IL-6 in sera samples of these mice was extremely high (15,479.9 ± 532 Pg/mL). Furthermore, these mice died in 24h of post infection compared to 30 h in the weak biofilm forming NT S. aureus infected group. The study demonstrates no association between the strength of PIA (polysaccharide intercellular adhesion)-dependent biofilm production by clinical NT S. aureus and mammary gland pathology in a mouse mastitis model. However, the role of biofilm in the virulence of S. aureus advancing the time of mortality in mice warrants further investigation.

Cuminum cyminum L. Essential Oil: A Promising Antibacterial and Antivirulence Agent Against Multidrug-Resistant Staphylococcus aureus.

Cuminum cyminum L. (cumin) is valued for its aromatic and medicinal properties. There are several reports of antibacterial activity of C. cyminum essential oil (CcEO). Accordingly, the present study was conducted to investigate the mechanism(s) of action of the CcEO against multidrug-resistant (MDR) Staphylococcus aureus. Therefore, 10 S. aureus MDR isolates, obtained from different sources, were selected based on the antibiotic susceptibility patterns and the Clinical and Laboratory Standards Institute definition and subjected to the examinations. Our results exhibited promising bacteriostatic and bactericidal properties of the CcEO. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration values ranged from 5 to 10 and 10 to 20 μL ⋅ mL–1, respectively. Scanning electron microscope was used to assess the bacterial cell structure and morphology after the induction with 1/2 MIC concentration of the CcEO. The observed morphological changes appeared to be deformation of the cell membrane and destruction of the cells. In the case of quorum sensing inhibitory potential, treatment of S. aureus isolates with the sub-MIC concentrations (1/2 MIC) of the CcEO significantly reduced the hld expression (3.13-fold downregulation), which considerably controls S. aureus quorum-sensing accessory regulator system. Another virulence factor influenced by the CcEO was the polysaccharide intercellular adhesion production system, as an important component of cell–cell adhesion and biofilm formation. Consequently, the expression level of the intercellular adhesion (ica) locus in the S. aureus cells was examined following treatment with CcEO. The results showed significant decrease (−3.3-fold) in ica expression, indicating that the CcEO could potentially interfere with the process of biofilm formation. Using the ethidium bromide efflux inhibition assay, the S. aureus NorA efflux pump was phenotypically but not genotypically (in quantitative polymerase chain reaction assay) affected by the CcEO treatment. Using gas chromatography–mass spectrometry analysis, cuminic aldehyde (38.26%), α,β-dihydroxyethylbenzene (29.16%), 2-caren-10-al (11.20%), and γ-terpinene (6.49%) were the most detected compounds. The antibacterial and antivirulence action of the CcEO at sub-MIC concentrations means that no microbial resistance will be promoted and developed after the treatment with this agent. These findings revealed that the CcEO is a promising antibacterial agent to control infections caused by the MDR S. aureus strains.

Effect of Tea Tree Oil on Methicillin-resistant Staphylococcus aureus Biofilm

This study on the effect of tea tree oil on the biofilm of Methicillin-resistant Staphylococcus aureus (MRSA) and its mechanism of action. Firstly, the MIC values of tea tree oil against MRSA and 12 clinically isolated strains were determined to be 0.08%~0.32%. It was found that 0.16% of tea tree oil had an inhibitory effect on the formation of MRSA biofilms. Tea tree oil at 0.32% could completely remove mature biofilms of MRSA while 0.16% could be significant reduce the mature biofilms by semi-quantitative crystal violet experiments and CLSM. Preliminary exploration of the biofilm mechanism found that tea tree oil could inhibit the secretion of polysaccharide intercellular adhesion (PIA) and extracellular DNA (eDNA), and the expression level of agr, ica, cid and sar during the formation of MRSA biofilm. Therefore, tea tree essential oil would have a strong anti-biofilm effect, and could inhibite the synthesis of PIA, the secretion of eDNA and the expression of genes related to biofilm formation.

Biofilm Formation and Multiplex PCR detection of icaABCD Operon in Staphylococcus capitis

Aims: The ability to form biofilm is a major virulence factor in the virulence of the Coagulase negative Staphylococcus (CoNS) group of bacteria. Being the most predominant member of CoNS, the ability of S. epidermidis in causing biofilm-associated infections has been well established. On the other hand, S. capitis and has always been regard as a non-pathogenic species although recently it was found to be responsible in a variety of infections. Hence, this study aimed to determine the biofilm formation capabilities and the presence of icaABCD genes in clinical isolates of S. capitis, which have emerged as an important opportunistic pathogen in clinical settings.
 Methodology: S. capitis was isolated and identified from 17 out of 200 clinical samples. Biofilm formation assay was performed quantitatively using a microtitre plate method. Mulitplex PCR primers for icaABCD genes were designed from DNA sequences coding for the icaA, B, C, and D structural genes of S capitis JF930147.1 which was compared together with five other species of Staphylococcus. Amplification of the icaABCD genes was performed using the designated primers.
 Results: From the 17 strains of S. capitis clinical isolates, 14 were identified as S. capitis subsp capitis while the remaining three were identified as S. capitis subsp ureolyticus. Except for two of the S. capitis subsp capitis isolates, the remaining strains were able to form biofilm, with majority of them were strong biofilm formers. Multiplex PCR was successful in amplifying the four icaABCD genes which was demonstrated in all the S. capitis isolates, including the two non-biofilm forming isolates.
 Conclusion: Majority of the S. capitis isolates were able to form biofilm phenotypically suggesting the possibility in causing opportunistic infections through indwelling medical devices. Multiplex PCR however was able to detect the presence of the icaABCD genes in all the S. capitis isolates. This suggests that the biofilm assessment on microtitre plate is not a definitive tool in determining the production of polysaccharide intercellular adhesion (PIA) but the production of the icaABCD genes could be a better assessment in determining biofilm production in Staphylococcus.

Antibacterial, Antibiofilm, and Antioxidant Activity of Polysaccharides Obtained from Fresh Sarcotesta of Ginkgo biloba: Bioactive Polysaccharide that Can Be Exploited as a Novel Biocontrol Agent.

Staphylococcus aureus (S. aureus) biofilm plays an important role in the persistence of chronic infection due to its resistance to antibiotics. Because of their functional diversity, active polysaccharide is increasingly being applied as a biocontrol agent to inhibit the formation of biofilm by pathogens. In this study, a new polysaccharide, GBSPII-1, isolated from the fresh sarcotesta of Ginkgo biloba L. (G. biloba) was characterized and its effect on antibiofilm formation of S. aureus was examined in vitro. High-Performance Liquid Chromatography (HPLC) analysis showed that GBSPII-1 is an acidic heteropolysaccharide composed of mannose, rhamnose, glucose, glucuronic acid, and galacturonic acid. GBSPII-1 demonstrated a molecular weight of 34 kDa and may affect the accumulation of polysaccharide intercellular adhesion (PIA) by inhibiting icaA, icaB, icaC, and icaD gene expression at subinhibitory concentrations. Under 10 g/L, GBSPII-1 showed an antioxidant effect on the inhibition rate of H2O2-induced erythrocyte hemolysis and the scavenging rate of DPPH radicals was 76.5 ± 0.5% and 89.2 ± 0.26%, respectively. The findings obtained in this study indicate that GBSPII-1 has antibacterial effect, is a possible source of natural antioxidants, and may be a potential biocontrol agent for the design of new therapeutic strategies for biofilm-related S. aureus infections.

Novel Strategies to Combat Bacterial Biofilms.

Biofilms are considered as a severe problem in the treatment of bacterial infections; their development causes some noticeable resistance to antibacterial agents. Biofilms are responsible for at least two-thirds of all infections, displaying promoted resistance to classical antibiotic treatments. Therefore, finding new alternative therapeutic approaches is essential for the treatment and inhibition of biofilm-related infections. Therefore, this review aims to describe the potential therapeutic strategies that can inhibit bacterial biofilm development; these include the usage of antiadhesion agents, AMPs, bacteriophages, QSIs, aptamers, NPs and PNAs, which can prevent or eradicate the formation of biofilms. These antibiofilm agents represent a promising therapeutic target in the treatment of biofilm infections and development of a strong capability to interfere with different phases of the biofilm development, including adherence, polysaccharide intercellular adhesion (PIA), quorum sensing molecules and cell-to-cell connection, bacterial aggregation, planktonic bacteria killing and host-immune response modulation. In addition, these components, in combination with antibiotics, can lead to the development of some kind of powerful combined therapy against bacterial biofilm-related infections.

Zinc- and strontium- co-incorporated nanorods on titanium surfaces with favorable material property, osteogenesis, and enhanced antibacterial activity.

Early infection and peri-implantitis after implant restoration are major reasons for dental implant failure. Implant-associated infections are majorly attributed to biofilm formation. In this study, co-incorporated zinc- (Zn-) and strontium- (Sr-) nanorod coating on sandblasted and acid-etched (SLA) titanium (SLA-Zn/Sr) was fabricated by hydrothermal synthesis. It was aimed at promoting osteogenesis while inhibiting biofilm formation. The nanorod-like particles (φ 30-50 nm) were found to be evenly formed on SLA-Zn/Sr (Zn: 1.49 ± 0.16 wt%; Sr: 21.69 ± 2.74 wt%) that was composed of well-crystallized ZnTiO3 and SrTiO3 phases. With a sufficient interface bonding strength (42.00 ± 3.00 MPa), SLA-Zn/Sr enhanced the corrosion resistance property of titanium. Besides, SLA-Zn/Sr promoted the cellular initial adhesion, proliferation and osteogenic differentiation of rBMSCs in vitro while inhibiting the adhesion of Staphylococcus aureus and Porphyromonas gingivalis . In addition, through down-regulating icaA gene expression, this novel surface reduced the secretion of polysaccharide intercellular adhesion (reduced by 87.9% compared to SLActive) to suppress the S. aureus biofilm formation. We, therefore, propose a new chemical modification on titanium for multifunctional implant material development. Due to the Zn/Sr co-doping in coating, material properties, early osteogenic effect and antibacterial ability of titanium can be simultaneously enhanced, which has the potential to be applied in dental implantation in the future.

PIA and rSesC Mixture Arisen Antibodies Could Inhibit the Biofilm-Formation in Staphylococcus aureus.

Staphylococcus aureus as a causative agent of hospital-acquired infections has been considered as the primary concern in biomaterial-related infections (BAIs). Following the purification of polysaccharide intercellular adhesion (PIA) as an efficient macromolecule in biofilm formation in the native condition, recombinant S. epidermidis surface-exposed rSesC protein, with the most homology to clumping factor A (ClfA) in S. aureus was cloned and expressed in a prokaryotic host as well. Fourier transform infrared spectrometry (FTIR) and Western blotting procedure analyzed purified PIA and protein, respectively. Then, the immune response was evaluated by measuring total IgG titers. Moreover, the capacity of Anti-biofilm forming activity of arisen antibodies to a biofilm-forming S. aureus strains was assessed by the semi-quantitative micro-plate procedure. Data showed that the total IgGs were boosted in mice immunized sera. By performing an inhibition assay, the biofilm inhibitory effect of secreted antibodies to test strain was observed. Arisen antibodies against the mixture significantly were more potent than PIA and rSesC, when comparing individual antigens in a biofilm inhibition assay. immunization of mice with mentioned antigens especially a mixture of them, could eliminate the biofilm formation process in S. aureus. Hopefully, this study corresponds to the suggestion that the immunization of mice with PIA and rSesC candidate vaccines could protect against S. aureus infection.

Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

Staphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.

Antibiofilm Activity of Small-Molecule ZY-214-4 Against Staphylococcus aureus

Staphylococcus aureus is the most important pathogenic bacteria in humans. As the resistance of S. aureus to existing antibiotics is increasing, there is an urgent need for new anti-infective drugs. S. aureus biofilms cause persistent infections and resist complete eradication with antibiotic therapy. The present study investigated the inhibitory effect of the novel small-molecule ZY-214-4 (C19H11BrNO4) on S. aureus biofilm formation. At a subinhibitory concentration (4 μg/ml), ZY-214-4 had no effect on the growth of S. aureus strains and also showed no cytotoxicity in human normal bronchial epithelial cells (Bease-2B). The results of a semi-quantitative biofilm test showed that ZY-214-4 prevented S. aureus biofilm formation, which was confirmed by scanning electron microscopy and confocal laser scanning microscopy. ZY-214-4 significantly suppressed the production of polysaccharide intercellular adhesion and prevented cell aggregation, and also inhibited the mRNA expression of icaA and other biofilm-related genes (eno, clfA/B, fnbB, fib, ebpS, psmα, and psmβ) in clinical S. aureus isolates. Thus, at a subinhibitory concentration, ZY-214-4 inhibits biofilm formation by preventing cell aggregation, highlighting its clinical potential for preventing or treating S. aureus infections.

Diclofenac May Induce PIA-Independent Biofilm Formation in Staphylococcus aureus Strains

Staphylococcus aureus is a pathogen commonly resistant to antibiotics. Biofilm formation is one of the important factors related to its virulence. Non-antibiotics drugs, such as nonsteroidal anti-inflammatory agents (NSAIDs), have been studied as an alternative for treating infections by multiresistant pathogens and biofilm-associated infections. In this study, the effects of NSAID sodium diclofenac on growth inhibition and biofilm formation of S. aureus were evaluated. The minimum inhibitory concentration (MIC) of diclofenac for fifty isolates ranged from 200 to 400 μg/mL. Diclofenac sub-MICs induced biofilm in 32.3% of biofilm-negative strains in tryptic soy broth. All biofilms induced by the drug showed a PIA- (polysaccharide intercellular adhesion-) independent composition, and the scanning electron microscopy showed that the induced biofilm presented a very discrete matrix. The combination of diclofenac with rifampicin sub-MICs induced strong production of PIA-dependent biofilm in three of four strains, while combination of NSAID with NaCl induced the formation of partially polysaccharide biofilm in two strains and PIA-independent biofilm in another strain. The combination of NSAID with glucose resulted in PIA-independent biofilms in all four strains tested. The results showed that diclofenac can commonly induce biofilm production by a PIA-independent pathway. However, when this NSAID is combined with other types of inducing agents, the composition of the biofilm produced may vary.

IgY Targeting Bacterial Quorum-Sensing Molecules in Implant-Associated Infections

Background: Implant-associated infections are still a major complication in the field of orthopedics. Bacteria can form biofilms on implant surfaces, making them more difficult to detect and treat. Since standard antibiotic therapy is often impaired in biofilm infections, particular interest is directed towards finding treatment alternatives. Biofilm-formation is a well-organized process during which bacteria communicate via quorum-sensing molecules (QSM). The aim of this study was to inhibit bacterial communication by directing avian IgY against specific QSM. Methods: Chicken were immunized against the following QSM: (1) AtlE, a member of the autolysin family which mediates attachment to a surface in Staphylococcus epidermidis; (2) GroEL, the bacterial heat shock protein; (3) PIA (polysaccharide intercellular adhesion), which is essential for cell–cell adhesion in biofilms. Staphylococcus epidermidis biofilms were grown and inhibition of biofilm-formation by IgYs was evaluated. Additionally, human osteoblasts were cultivated and biocompatibility of IgYs was tested. Results: We were able to demonstrate that all IgYs reduced biofilm-formation, also without prior immunization. Therefore, the response was probably not specific with regard to the QSM. Osteoblasts were activated by all IgYs which was demonstrated by microscopy and an increased release of IL-8. Conclusions: In conclusion, avian IgY inhibits biofilm-formation, though the underlying mechanism is not yet clear. However, adverse effects on local tissue cells (osteoblasts) were also observed.