Exploring differentially expressed genes of Staphylococcus aureus exposed to human tonsillar cells using RNA sequencing

Background - The nose and the throat are the most predominant colonizing sites of Staphylococcus aureus, and colonization is a risk factor for infection. Nasal colonization is well described; however, we have limited knowledge about S. aureus throat colonization. The main objective of this study was to explore differentially expressed genes (DEGs) in S. aureus throat isolate TR145 exposed to human tonsil epithelial cells (HTEpiC) by using RNA sequencing (RNA-seq) and pathway analysis. DEGs in S. aureus at 1 or 3 hours (h) interaction with its host were explored. Results - S. aureus was co-cultured in absence and presence of tonsillar cells at 1 or 3 h. Over the 3 h time frame, the bacteria multiplied, but still caused only minor cytotoxicity. Upon exposure to tonsillar cell line, S. aureus changed its transcriptomic profile. A total of 508 DEGs were identified including unique (1 h, 160 DEGs and 3 h, 78 DEGs) and commonly shared genes (1 and 3 h, 270 DEGs). Among the DEGs, were genes encoding proteins involved in adhesion and immune evasion, as well as iron acquisition and transport. Reverse transcription qPCR was done on selected genes, and the results correlated with the RNA-seq data. Conclusion - We have shown the suitability of using HTEpiC as an in vitro model for investigating key determinants in S. aureus during co-incubation with host cells. Several DEGs were unique after 1 or 3 h exposure to host cells, while others were commonly expressed at both time points. As their expression is induced upon meeting with the host, they might be explored further for future targets for intervention to prevent either colonization or infection in the throat

Liposomal delivery of antibiotic loaded nucleic acid nanogels with enhanced drug loading and synergistic anti-inflammatory activity against S. aureus intracellular infections

The persistence of Staphylococcus aureus has been accredited to its ability to escape immune response via host cell invasion. Despite the efficacy of many antibiotics against S. aureus, the high extracellular concentrations of conventional antibiotics required for bactericidal activity is limited by their low cellular accumulation and poor intracellular retention. While nanocarriers have received tremendous attention for antibiotic delivery against persistent pathogens, they suffer daunting challenges such as low drug loading, poor retention and untimely release of hydrophilic cargos. Here, a hybrid system (Van_DNL) is fabricated wherein nucleic acid nanogels are caged within a liposomal vesicle for antibiotic delivery. The central principle of this approach relies on exploiting non-covalent electrostatic interactions between cationic cargos and polyanionic DNA to immobilize antibiotics and enable precise temporal release against intracellular S. aureus. In vitro characterization of Van_DNL revealed a stable homogenous formulation with circular morphology and enhanced vancomycin loading efficiency. The hybrid system significantly sustained the release of vancomycin over 24 h compared to liposomal or nanogel controls. Under enzymatic conditions relevant to S. aureus infections, lipase triggered release of vancomycin was observed from the hybrid. While using Van_DNL to treat S. aureus infected macrophages, a dose dependent reduction in intracellular bacterial load was observed over 24 h and exposure to Van_DNL for 48 h caused negligible cellular toxicity. Pre-treatment of macrophages with the antimicrobial hybrid resulted in a strong anti-inflammatory activity in synergy with vancomycin following endotoxin stimulation. Conceptually, these findings highlight these hybrids as a unique and universal platform for synergistic antimicrobial and anti-inflammatory therapy against persistent infections

Chitosan-based delivery system enhances antimicrobial activity of chlorhexidine

Infected chronic skin wounds and other skin infections are increasingly putting pressure on the health care providers and patients. The pressure is especially concerning due to the rise of antimicrobial resistance and biofilm-producing bacteria that further impair treatment success. Therefore, innovative strategies for wound healing and bacterial eradication are urgently needed; utilization of materials with inherent biological properties could offer a potential solution. Chitosan is one of the most frequently used polymers in delivery systems. This bioactive polymer is often regarded as an attractive constituent in delivery systems due to its inherent antimicrobial, anti-inflammatory, anti-oxidative, and wound healing properties. However, lipid-based vesicles and liposomes are generally considered more suitable as delivery systems for skin due to their ability to interact with the skin structure and provide prolonged release, protect the antimicrobial compound, and allow high local concentrations at the infected site. To take advantage of the beneficial attributes of the lipid-based vesicles and chitosan, these components can be combined into chitosan-containing liposomes or chitosomes and chitosan-coated liposomes. These systems have previously been investigated for use in wound therapy; however, their potential in infected wounds is not fully investigated. In this study, we aimed to investigate whether both the chitosan-containing and chitosan-coated liposomes tailored for infected wounds could improve the antimicrobial activity of the membrane-active antimicrobial chlorhexidine, while assuring both the anti-inflammatory activity and cell compatibility. Chlorhexidine was incorporated into three different vesicles, namely plain (chitosan-free), chitosan-containing and chitosan-coated liposomes that were optimized for skin wounds. Their release profile, antimicrobial activities, anti-inflammatory properties, and cell compatibility were assessed in vitro. The vesicles comprising chitosan demonstrated slower release rate of chlorhexidine and high cell compatibility. Additionally, the inflammatory responses in murine macrophages treated with these vesicles were reduced by about 60% compared to non-treated cells. Finally, liposomes containing both chitosan and chlorhexidine demonstrated the strongest antibacterial effect against Staphylococcus aureus. Both chitosan-containing and chitosan-coated liposomes comprising chlorhexidine could serve as excellent platforms for the delivery of membrane-active antimicrobials to infected wounds as confirmed by improved antimicrobial performance of chlorhexidine

Polymyxin B stabilized DNA micelles for sustained antibacterial and antibiofilm activity against P. aeruginosa

Nucleic acid-based materials showcase an increasing potential for antimicrobial drug delivery. Although numerous reports on drug-loaded DNA nanoparticles outline their pivotal antibacterial activities, their potential as drug delivery systems against bacterial biofilms awaits further studies. Among different oligonucleotide structures, micellar nanocarriers derived from amphiphilic DNA strands are of particular interest due to their spontaneous self-assembly and high biocompatibility. However, their clinical use is hampered by structural instability upon cation depletion. In this work, we used a cationic amphiphilic antibiotic (polymyxin B) to stabilize DNA micelles destined to penetrate P. aeruginosa biofilms and exhibit antibacterial/antibiofilm properties. Our study highlights how the strong affinity of this antibiotic enhances the stability of the micelles and confirms that antibacterial activity of the novel micelles remains intact. Additionally, we show that PMB micelles can penetrate P. aeruginosa biofilms and impact their metabolic activity. Finally, PMB micelles were highly safe and biocompatible, highlighting their possible application against P. aeruginosa biofilmcolonized skin wounds

Co-culturing with Streptococcus anginosus alters Staphylococcus aureus transcriptome when exposed to tonsillar cells

IntroductionImproved understanding of Staphylococcus aureus throat colonization in the presence of other co-existing microbes is important for mapping S. aureus adaptation to the human throat, and recurrence of infection. Here, we explore the responses triggered by the encounter between two common throat bacteria, S. aureus and Streptococcus anginosus, to identify genes in S. aureus that are important for colonization in the presence of human tonsillar epithelial cells and S. anginosus, and further compare this transcriptome with the genes expressed in S. aureus as only bacterium.MethodsWe performed an in vitro co-culture experiment followed by RNA sequencing to identify interaction-induced transcriptional alterations and differentially expressed genes (DEGs), followed by gene enrichment analysis.Results and discussionA total of 332 and 279 significantly differentially expressed genes with p-value < 0.05 and log2 FoldChange (log2FC) ≥ |2| were identified in S. aureus after 1 h and 3 h co-culturing, respectively. Alterations in expression of various S. aureus survival factors were observed when co-cultured with S. anginosus and tonsillar cells. The serine-aspartate repeat-containing protein D (sdrD) involved in adhesion, was for example highly upregulated in S. aureus during co-culturing with S. anginosus compared to S. aureus grown in the absence of S. anginosus, especially at 3 h. Several virulence genes encoding secreted proteins were also highly upregulated only when S. aureus was co-cultured with S. anginosus and tonsillar cells, and iron does not appear to be a limiting factor in this environment. These findings may be useful for the development of interventions against S. aureus throat colonization and could be further investigated to decipher the roles of the identified genes in the host immune response in context of a throat commensal landscape

Infectious pancreatic necrosis virus (IPNV) - Persistent infections, virulence and antiviral defence

Infeksiøs pankreas nekrose (IPN) er en fiskesykdom som rammer mange fiskeslag over store deler av verden. Det har store økonomiske konsekvenser for norsk lakseoppdrettsnæring. Infeksiøs pankreas nekrose var først kjent som en sykdom i yngel, men i de siste 10 til 20 årene har det vært et like stort problem i post-smolt. Antall utbrudd varierer fra år til år, der 2009 ble registrert som et år med flest utbrudd. Samme år introduserte avlsselskapet AquaGen QTL-rogn. Denne fisken var svært motstandsdyktig mot IPN. Sykdomsproblemene forsvant i de anleggene som benyttet seg av IPN-QTL-rogn, noe som gjenspeiler seg i at det har vært en drastisk nedgang i antall rapporterte sykdomsutbrudd i norsk lakseoppdrettsnæring. Resultatet av et IPN utbrudd varierer mye, alt fra lav til høy dødelighet. Mye av denne variasjonen er knyttet til de forskjellige virusisolaters sykdomsfremkallende (virulens) egenskaper. Denne avhandlingen beskriver hvordan molekylære forskjeller i feltisolater er med på å gi virusene forskjellige virulensegenskaper. Ved å samle inn feltisolater, bestemme nukleotidsekvensen til deler av VP2-genet til de forskjellige isolatene og utføre smitteforsøk med de ulike virusisolatene har vi vist at spesifikke aminosyrer er med på å bestemme virusisolatets virulensegenskaper. Fisk som er bærer av IPNV er symptomfrie med lavt eller ikke detekterbart nivå av virus. Det var derfor nødvendig å utvikle en metode for detektering av virus som var mer sensitiv enn den som tidligere var brukt. En real-time PCR for detektering av transkriptet av VP2-genet ble derfor utviklet. Referansegener ble validert og metoden ble optimalisert for å gi en metode med høy sensitivitet og nøyaktighet. Når laksen møter IPNV for første gang er resultatet av infeksjonen avhengig både av virulensegenskaper hos viruset og hvor effektivt det medfødte antivirale immunforsvaret til fisken er. Makrofager er viktige celler i det medfødte immunforsvaret, men de representerer også et reservoar for mange persisterende virus, deriblant IPNV. I dette arbeidet har vi smittet smolt i ferskvannsfasen med virus med høy og lav virulens. Vi har sett på forskjeller i utviklingen av sykdom mellom virus isolatene ved å måle mengde virus og se på dødelighet i sjøfasen. Vi har også undersøkt om det antivirale forsvaret til laksen reagerer forskjellig mot de ulike virusisolatene

The Antimicrobial Properties of Chitosan Can be Tailored by Formulation

Topical administration of drugs into the vagina can provide local therapy of vaginal infections, preventing the possible systemic side effects of the drugs. The natural polysaccharide chitosan is known for its excellent mucoadhesive properties, safety profile, and antibacterial effects, and thus it can be utilized in improving localized vaginal therapy by prolonging the residence time of a drug at the vaginal site while acting as an antimicrobial in synergy. Therefore, we aimed to explore the potential of chitosan, namely chitosan-coated liposomes and chitosan hydrogel, as an excipient with intrinsic antimicrobial properties. Liposomes were prepared by the thin-film hydration method followed by vesicle size reduction by sonication to the desired size, approximately 200 nm, and coated with chitosan (0.01, 0.03, 0.1, and 0.3%, w/v, respectively). The mucoadhesive properties of chitosan-coated liposomes were determined through their binding efficiency to mucin compared to non-coated liposomes. Non-coated liposomal suspensions were incorporated in chitosan hydrogels forming the liposomes-in-hydrogel formulations, which were further assessed for their texture properties in the presence of biological fluid simulants. The antibacterial effect of chitosan-coated liposomes (0.03%, 0.1% and 0.3%, w/v) and chitosan hydrogels (0.1% and 0.3%, w/w) on Staphylococcus epidermidis and Staphylococcus aureus was successfully confirmed

Biofilm Responsive Zwitterionic Antimicrobial Nanoparticles to Treat Cutaneous Infection

To avert the poor bioavailability of antibiotics during S. aureus biofilm infections, a series of zwitterionic nanoparticles containing nucleic acid nanostructures were fabricated for the delivery of vancomycin. The nanoparticles were prepared with three main lipids: (i) neutral (soy phosphatidylcholine; P), (ii) positively charged ionizable (1,2-dioleyloxy-3-dimethylaminopropane; D), and (iii) anionic (1,2-dipalmitoyl-sn-glycero-3-phospho((ethyl-1′,2′,3′-triazole) triethylene glycolmannose; M) or (cholesteryl hemisuccinate; C) lipids. The ratio of the anionic lipid was tuned between 0 and 10 mol %, and its impact on surface charge, size, stability, toxicity, and biofilm sensitivity was evaluated. Under biofilm mimicking conditions, the enzyme degradability (via dynamic light scattering (DLS)), antitoxin (via DLS and spectrophotometry), and antibiotic release profile was assessed. Additionally, biofilm penetration, prevention (in vitro), and eradication (ex vivo) of the vancomycin loaded formulation was investigated. Compared with the unmodified nanoparticles which exhibited the smallest size (188 nm), all three surface modified formulations showed significantly larger sizes (i.e., 222–277 nm). Under simulations of biofilm pH conditions, the mannose modified nanoparticle (PDM 90/5/5) displayed ideal charge reversal from a neutral (+1.69 ± 1.83 mV) to a cationic surface potential (+17.18 ± 2.16 mV) to improve bacteria binding and biofilm penetration. In the presence of relevant bacterial enzymes, the carrier rapidly released the DNA nanoparticles to function as an antitoxin against α-hemolysin. Controlled release of vancomycin prevented biofilm attachment and significantly reduced early stage biofilm formations within 24 h. Enhanced biocompatibility and significant ex vivo potency of the PDM 90/5/5 formulation was also observed. Taken together, these results emphasize the benefit of these nanocarriers as potential therapies against biofilm infections and fills the gap for multifunctional nanocarriers that prevent biofilm infections

Chitosomes-In-Chitosan Hydrogel for Acute Skin Injuries: Prevention and Infection Control

Burns and other skin injuries are growing concerns as well as challenges in an era of antimicrobial resistance. Novel treatment options to improve the prevention and eradication of infectious skin biofilm-producing pathogens, while enhancing wound healing, are urgently needed for the timely treatment of infection-prone injuries. Treatment of acute skin injuries requires tailoring of formulation to assure both proper skin retention and the appropriate release of incorporated antimicrobials. The challenge remains to formulate antimicrobials with low water solubility, which often requires carriers as the primary vehicle, followed by a secondary skin-friendly vehicle. We focused on widely used chlorhexidine formulated in the chitosan-infused nanocarriers, chitosomes, incorporated into chitosan hydrogel for improved treatment of skin injuries. To prove our hypothesis, lipid nanocarriers and chitosan-comprising nanocarriers (≈250 nm) with membrane-active antimicrobial chlorhexidine were optimized and incorporated into chitosan hydrogel. The biological and antibacterial effects of both vesicles and a vesicles-in-hydrogel system were evaluated. The chitosomes-in-chitosan hydrogel formulation demonstrated promising physical properties and were proven safe. Additionally, the chitosan-based systems, both chitosomes and chitosan hydrogel, showed an improved antimicrobial effect against S. aureus and S. epidermidis compared to the formulations without chitosan. The novel formulation could serve as a foundation for infection prevention and bacterial eradication in acute wounds