Expression Of Virulence Factors Research Articles

Establishing Reference Genes for Accurate Gene Expression Profiling in Toxigenic Bacillus cereus

Bacillus cereus is a Gram-positive pathogen associated with foodborne illnesses and severe non-gastrointestinal infections. Robust tools for accurate gene transcription analysis are essential for studying toxin gene expression dynamics and deciphering the complex regulatory networks orchestrating the expression of toxin and virulence factors. This study aimed to identify reliable reference genes for normalizing reverse transcription quantitative PCR (RT-qPCR) data in toxigenic B. cereus. An emetic and an enteropathogenic strain were used as model organisms to establish a suitable reference gene set to monitor the dynamics of toxin gene transcription. Ten candidate reference genes were evaluated for their expression stability using geNorm, NormFinder, BestKeeper and the ΔCq method, with the final rankings integrated via RefFinder. Among the tested genes, rho, rpoD and recA were identified as the most stable expressed reference genes across all tested conditions. As shown in this proof-of-principle study, the established reference gene set provides a suitable tool to investigate the influence of extrinsic and intrinsic factors on toxin gene transcription. In conclusion, our newly established reference gene set provides a robust basis for studying toxin gene expression in B. cereus and contributes to a better understanding of its pathogenicity and potential strategies to mitigate its harmful effects.

Repurposing Diflunisal as an Anti-Virulence Agent Against Staphylococcus aureus

Abstract Infections refractory to standard antibiotic therapy are contributing to adverse treatment outcomes in patients suffering from deep-seated bacterial infections caused by increasingly resistant pathogens. Adjunctive strategies targeting bacterial virulence factors have been considered to supplement the host immune response in fighting the infection. Previous studies suggest that the FDA-approved anti-inflammatory drug diflunisal inhibits Staphylococcus aureus (SA) α-toxin expression by its interaction with the response regulator AgrA. We investigated the broader anti-virulence properties of diflunisal against pathogenic strains of SA and established proof-of-concept for its efficacy in blocking SA virulence. Our studies reveal that diflunisal inhibits α-toxin production, sensitizes SA to cationic antibiotics and human antimicrobial peptides, inhibits the production of the golden pigment staphyloxanthin, and reduces biofilm formation. Molecular docking simulations revealed potential interactions between diflunisal and AgrA binding sites. In addition, sequence alignment of the SA AgrA response regulator demonstrated similarities to other response regulators involved in controlling virulence factor expression. Appreciation of the antivirulence properties of diflunisal supports a therapeutic strategy distinct from structurally similar compounds, such as salicylic acid. The repurposing of diflunisal may mitigate disease severity and provide a unique adjunctive tool in the treatment of SA infection.

Aloin remodels the cell wall of Candida albicans to reduce its hyphal virulence against oral candidiasis

Aloe vera (L.) Burm.f. is a traditional Chinese medicine known for treating various ailments, including fungal infections. Aloin is one of the major components from A. vera, but its antifungal mechanism and therapeutic potential against oral candidiasis are not clear. This study aimed to examine the mechanism of aloin against Candida albicans and its inhibitory activity against oral candidiasis. In this study, we for the first time found that aloin could induce the formation of abnormal hyphae with smaller hyphal diameters and fewer branching points in C. albicans including 11 clinical isolates without growth inhibition. The transcriptome and further cell wall contents analysis indicated that aloin remodeled the cell wall to increase the contents of β-1,3-glucan and furtherly showed an antagonistic effect with micafungin. Aloin also significantly inhibited the cell damage of oral epithelial cells and oral candidiasis in mice infected by C. albicans due to its inhibitory actions on the hyphal development and expressions of virulence factors, including candidalysin (coded by ECE1). Our results suggest that aloin is a promising antifungal agent for controlling candidiasis and targeting hyphal development and pathogenesis represents a practical strategy for developing new antifungal drugs.Key points• Aloin remodels the C. albicans cell wall to form avirulent hyphae.• Aloin inhibits C. albicans infections in oral epithelial cells and mouse mucosa without toxicity.• Aloin is a promising antifungal agent with therapeutic potential against C. albicans infections.Graphical

Mesalazine: a novel therapeutic agent for periodontitis via regulation of periodontal microbiota and inhibiting Porphyromonas gingivalis

IntroductionPeriodontitis and inflammatory bowel disease are chronic inflammatory diseases with shared epidemiological, biological, and therapeutic associations. Given the similarities in their pathogenic factors, this study hypothesized that mesalazine, a key treatment agent for inflammatory bowel disease, could also be effective in managing periodontitis.MethodsThe antimicrobial effect of mesalazine on Porphyromonas gingivalis was investigated in vitro, including observations of morphological changes on the surface of P. gingivalis. Additionally, the impact of mesalazine on both the formation and established plaque biofilms was examined. The antimicrobial mechanism was elucidated by assessing the expression of P. gingivalis virulence genes and by determining the disruptive effect on P. gingivalis cell membranes. An in vivo rat model of periodontitis was constructed to evaluate mesalazine’s efficacy and its influence on the periodontal bacterial flora in the context of periodontitis.Results and discussionOur results demonstrated that mesalazine concentrations ranging from 0.5 to 2 mg/mL significantly inhibited P. gingivalis proliferation over 72 h. Flow cytometry revealed a marked reduction in the number of viable cells following mesalazine treatment. At the nanometer scale, mesalazine induced crumpling and rupture of the P. gingivalis surface, compromising cell membrane integrity. Mesalazine not only suppressed the formation of plaque biofilms by P. gingivalis and polymicrobial communities but also disrupted pre-existing biofilms. The data also suggested that mesalazine could disrupt the integrity of the P. gingivalis cell membrane and inhibit the expression of virulence factors. An animal model of periodontitis in rats was successfully constructed in vivo. Mesalazine treatment inhibited alveolar bone resorption, alleviated inflammation of periodontal tissues, and improved the composition of the periodontal flora to a healthier state. This study establishes that mesalazine can treat periodontitis through modulation of the periodontal flora and its anti-inflammatory properties, thus broadening its classical therapeutic applications.

Dual RNA sequencing of a co-culture model of Pseudomonas aeruginosa and human 2D upper airway organoids

Pseudomonas aeruginosa is a Gram-negative bacterium that is notorious for airway infections in cystic fibrosis (CF) subjects. Bacterial quorum sensing (QS) coordinates virulence factor expression and biofilm formation at population level. Better understanding of QS in the bacterium-host interaction is required. Here, we set up a new P. aeruginosa infection model, using 2D upper airway nasal organoids that were derived from 3D organoids. Using dual RNA-sequencing, we dissected the interaction between organoid epithelial cells and WT or QS-mutant P. aeruginosa strains. Since only a single healthy individual and a single CF subject were used as donors for the organoids, conclusions about CF-specific effects could not be deduced. However, P. aeruginosa induced epithelial inflammation, whereas QS signaling did not affect the epithelial airway cells. Conversely, the epithelium influenced infection-related processes of P. aeruginosa, including QS-mediated regulation. Comparison of our model with samples from the airways of CF subjects indicated that our model recapitulates important aspects of infection in vivo. Hence, the 2D airway organoid infection model is relevant and may help to reduce the future burden of P. aeruginosa infections in CF.

Correlation Analysis Between Multi-Drug Resistance Phenotype and Virulence Factor Expression of Clinical Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA), as a common pathogen of nosocomial infections, has been experiencing an increasing rate of drug resistance with the widespread use and abuse of antimicrobial drugs. High-drug-resistance and high-virulence phenotypes are two distinctive features of the strong pathogenicity of multi-drug-resistant PA. Exploring the characterization of virulence factor expression and its relationship with the multi-drug resistance phenotype is essential to reduce the further development of resistance as well as a high standard of infection prevention and control. A total of 50 PA isolated from clinical practice were collected. The Kirby-Bauer test was used for drug-sensitive screening, and the results showed that 16 strains were resistant and 16 strains were sensitive. The drug resistance rate of multi-drug-resistant PA against cefepime, cefazolin, ampicillin, and imipenem was up to 100%. The multi-drug-resistant groups were superior in producing pyocyanin and forming biofilm to the sensitive groups. The distribution of isolates with different swarming motility capacities and elastase levels did not show pronounced differences among the multi-drug-resistant and sensitive groups. In addition, biofilm formation was moderately associated with imipenem resistance. Among the strains with strong virulence factor expression, the gene bands showed little difference, suggesting that the gene is highly homologous. The virulence factor matrix analysis showed that there were different degrees of correlation among the 4 virulence factors. The correlation between multidrug-resistant PA and virulence factor expression is complex. PA, which were good at producing pyocyain and forming biofilm, were highly resistant to cephalosporins, beta-lactams and carbepenems; hence, such drugs are not proper for anti-infective treatment in clinics.

A strategy for controlling Hypervirulent Klebsiella pneumoniae: inhibition of ClpV expression

The emergence and prevalence of hypervirulent Klebsiella pneumoniae (hvKP) have proposed a great challenge to control this infection. Therefore, exploring some new drugs or strategies for treating hvKP infection is an urgent issue for scientific researchers. In the present study, the clpV gene deletion strain of hvKP (ΔclpV-hvKP) was constructed using CRISPR-Cas9 technology, and the biological characteristics of ΔclpV-hvKP were investigated to explore the new targets for controlling this pathogen. The results showed that clpV gene deletion did not affect the growth ability of hvKP. However, knocking out the clpV gene markedly decreased the mucoid phenotype and the biofilm formation ability of hvKP. It reduced the interspecific competition of hvKP with Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus. The clpV deletion significantly changed the transcriptome profile of hvKP, inhibited the expression of virulence factors, and decreased the lethality of hvKP against Galleria mellonella larvae. In vitro experiments showed that lithocholic acid could inhibit the expression of the clpV gene and reduce the virulence of hvKP. Our data suggested that the clpV gene may be a potential target for decreasing hvKP infection risk.

Sesamin targets ClpP which attenuates virulence of S. aureus and protects mice from fatal pneumonia induced by MRSA.

The aim of this study was to identify sesamin as a Casein hydrolase P (ClpP) inhibitor and to determine whether it could attenuate the virulence of methicillin-resistant Staphylococcus aureus (MRSA). Through fluorescence resonance energy transfer (FRET) screening, a natural compound sesamin demonstrated a significant inhibitory effect on ClpP enzyme activity with an IC50 of 20.62μg/mL. Sesamin suppressed the expression of virulence factors of MRSA such as α-hemolysin (Hla) and Panton-Valentine leucocidin (PVL) by protein immunoblotting. Thermal shift assay (TSA) and cellular thermal shift assay (CETSA) showed that sesamin could bind to ClpP and enhance the thermal stability of ClpP. Furthermore, the binding affinity between sesamin and ClpP was determined by surface plasmon resonance (SPR) with a KD value of 7.18×10-6M. Molecular docking, dynamics simulations and point mutation analysis confirmed the stability of the sesamin-ClpP complex with a -10.184kcal/mol total binding energy and identified PHE-174 in ClpP as a key binding site. In mice pneumonia model, sesamin combined vancomycin treatment markedly reduced the pathogenicity of MRSA-infected mice, offering protection against fatal lung infections. Overall, these findings validate sesamin as a promising compound that targets ClpP, reducing virulence factor expression, that holds potential as a hit compound against MRSA infections.

Exploring the antivirulence potential of phenolic compounds to inhibit quorum sensing in Pseudomonas aeruginosa.

Bacteria coordinate gene expression in a cell density-dependent manner in a communication process called quorum sensing (QS). The expression of virulence factors, biofilm formation and enzyme production are QS-regulated phenotypes that can interfere in human health. Due to this importance, there is great interest in inhibiting QS, comprising an anti-virulence strategy. This work aimed to evaluate the effect of selected phenolic compounds on the inhibition of QS-regulated phenotypes in Pseudomonas aeruginosa PAO1, using concentrations that do not interfere in bacterial growth. This is one of the main premises for studying the effect of compounds on QS. Firstly, an in-silico study with the LasR and RhlR proteins of P. aeruginosa by molecular docking of 82 phenolic compounds was performed. Then, a screening with 13 selected phenolic compounds was performed, using biosensor strains P. aeruginosa lasB-gfp and P. aeruginosa rhlA-gfp, which emit fluorescence when the QS system is activated. From this assay, eight compounds were selected and evaluated for inhibition of pyocyanin, rhamnolipids, proteases, elastase, and motility. The compounds variably inhibited the evaluated virulence factors. The greatest inhibitions were observed for swarming motility, achieving inhibition rates of up to 50% for baicalein (500 µM) and curcumin (50 µM). Notably, curcumin showed satisfactory inhibition for all phenotypes even at lower concentrations (12.5 to 50 µM) compared to the other compounds (125 to 500 µM). Four compounds - rosmarinic acid, baicalein, curcumin, and resveratrol - were finally tested against biofilm formation observed by optical microscopy. This study demonstrated that phenolic compounds exhibit strong in silico binding to P. aeruginosa LasR and RhlR proteins and variably inhibit QS-regulated phenotypes in vitro. Although no biofilm inhibition was observed, future studies combining compounds and exploring molecular mechanisms are recommended. These findings highlight the biotechnological potential of phenolic compounds for future applications in the food, clinical, and pharmaceutical fields.

Comparative study of phenotypic and genotypic expression of virulence factors in colonizing and pathogenic carbapenem resistant Acinetobacter baumannii (CRAB)

Carbapenem resistant Acinetobacter baumannii has evolved as the most troublesome microorganism with multiple virulence factors. Biofilm formation, porins, micronutrient capturing mechanism and quorum sensing, provide protection against desiccation, host-pathogen killing and enhance its persistence. The conservation of these factors between colonizing and pathogenic carbapenem resistant A. baumannii has been barely investigated. We studied biofilm formation, desiccation survival, motility and hemolysis in pathogenic carbapenem resistant A. baumannii and colonizer carbapenem resistant A. baumannii from the hospital environment. The virulent genes pgaA, csuE, bap, ompA, abaI, pilA and bauA were detected by simplex-PCR and Quantitative Real-Time PCR was done for expressional studies. In-vivo survival percentage was studied by Galleria mellonella (wax moth) killing assay. Phenotypic characterization revealed that the biofilm formation and desiccation survival proportion was significantly higher in colonizer carbapenem resistant A. baumannii (p < 0.05). Twitching motility was found comparable (mean 0.5 to 1.5 cm). Surface associated motility varied widely. None showed hemolysis. The csuE, bap, ompA, abaI, pilA and bauA genes were detected in almost all the pathogenic and colonizer carbapenem resistant A. baumannii isolates while none harboured pgaA gene. The expression of bap, ompA and bauA gene was found significantly higher in pathogenic carbapenem resistant A. baumannii while expression of csuE and abaI gene was comparable in both. Overexpression of pilA gene was seen in those with higher surface associated motility. Pathogenic carbapenem resistant A. baumannii showed significantly higher pathogenicity in-vivo, as 100% of larvae died on 4th day post-infection. In conclusion high level expression of outer membrane proteins (ompA) and siderophores is significantly associated with the pathogenicity in carbapenem resistant A. baumannii isolated from infections, which can be a differentiating point from the colonizers.Clinical TrialNot Applicable

Bioorganic compounds in quorum sensing disruption: strategies, Mechanisms, and future prospects.

Recent research has shed light on the complex world of bacterial communication through quorum sensing. This sophisticated intercellular signalling mechanism, driven by auto-inducers, regulates crucial bacterial community behaviours such as biofilm formation, expression of virulence factors, and resistance mechanisms. The increasing threat of antibiotic resistance, coupled with quorum sensing mediated response, necessitates alternative strategies to combat bacterial infections. Quorum quenching has emerged as a promising approach, utilizing quorum quenching enzymes and quorum sensing inhibitors to disrupt quorum sensing signalling pathways, thus reducing virulence and biofilm formation. This review focuses on natural and synthetic bioorganic compounds that act as quorum-sensing inhibitors, providing insights into their mechanisms, structure-activity relationships, and potential as anti-virulence agents. The review also explores the communication languages of bacteria, including AHLs in gram-negative bacteria, oligopeptides in gram-positive bacteria, and LuxS, a universal microbial language. By highlighting recent advancements and prospects in bioorganic QSIs, this article underscores their crucial role in developing effective anti-virulence therapies and combating the growing threat of antimicrobial resistance.

Polypeptide of Inonotus hispidus extracts alleviates periodontitis through suppressing inflammatory bone loss

This study aimed to characterize and evaluate the effects of a novel polypeptide isolated from Inonotus hispidus (IH) against periodontitis. The polypeptides extracted and purified from the fruiting body of IH had a uniform molar mass, including 23 types of peptides. IH polypeptide (IHP) exerted antimicrobial activity against Porphyromonas gingivalis (P. gingivalis) by damaging the cell walls and membranes of microorganisms, disturbing energy metabolism, and regulating the expression of virulence factors. IHP significantly inhibited inflammation in lipopolysaccharides (LPS)-stimulated Raw264.7 cells evidenced by the regulation of inflammatory cytokine levels. In rats with ligature-induced periodontitis, IHP treatment ameliorated alveolar bone destruction and preserved the balance between oral flora and gut microbes. The interaction between oral and intestinal flora possibly affected the relevant metabolites. Proteomics combined with confirmation experiment revealed that the β-catenin/ nuclear factor-kappa B (NF-κB) signaling may be involved in IHP-mediated anti-periodontitis in rats, which helps reduce the secretion of pro-inflammatory factors and inhibit inflammatory osteoclastic response in the periodontal tissue. Additionally, IHP improved clinical parameters, including the plaque index (PLI), pocket depth (PD), bleeding on probing (BOP), and average probing depth in individuals with periodontitis. These findings augment the understanding of the potential role of IHP in treating periodontitis.

Inhibitory effects of berberine on fungal growth, biofilm formation, virulence, and drug resistance as an antifungal drug and adjuvant with prospects for future applications.

Berberine (BBR), an isoquinoline alkaloid found in medicinal plants such as Coptidis rhizoma, Berberis sp., and Hydrastis canadensis, is a distinctive compound known for its dual ability to exhibit broad-spectrum antifungal activity while offering beneficial effects to the host. These attributes make it a highly valuable candidate for antifungal therapy and as an antibiotic adjuvant. This review provides a comprehensive evaluation of BBR's antifungal properties, focusing on its in vitro and in vivo activity, underlying mechanisms, and its influence on fungal pathogenicity, including virulence, biofilm formation, and resistance. Additionally, the antifungal potential of BBR extracts, derivatives, and nanoformulations is examined in detail. BBR demonstrates fungicidal effects through multiple mechanisms. It targets critical fungal components such as mitochondria, cell membranes, and cell walls, while also inhibiting enzymatic activity and transcription processes. Furthermore, it suppresses the expression of virulence factors, effectively diminishing fungal pathogenicity. Beyond its direct antifungal activity, BBR exerts beneficial effects on the host by modulating gut microbiota, thereby bolstering host defenses against fungal infections and reducing potential adverse effects. BBR's interaction with conventional antifungal drugs presents a unique complexity, particularly in the context of resistance mechanisms. When used in combination therapies, conventional antifungal drugs enhance the intracellular accumulation of BBR, thereby amplifying its antifungal potency as the primary active agent. These synergistic effects position BBR as a promising candidate for combination strategies, especially in addressing drug-resistant fungal infections and persistent biofilms. As antifungal resistance and biofilm-associated infections continue to rise, the multifaceted properties of BBR and its advanced formulations highlight their significant therapeutic potential. However, the scarcity of robust in vivo and clinical studies limits a full understanding of its efficacy and safety profile. To bridge this gap, future investigations should prioritize well-designed in vivo and clinical trials to thoroughly evaluate the therapeutic effectiveness and safety of BBR in diverse clinical settings. This approach could pave the way for its broader application in combating fungal infections.

Silent signals: how N-acyl homoserine lactones drive oral microbial behaviour and health outcomes.

N-acyl homoserine lactones (AHLs) are small signalling molecules predominantly secreted in Gram-negative bacteria. The aim is to provide a comprehensive overview of AHLs in oral health. Two independent researchers conducted a systematic search of English language publications up to 30 June 2024 in PubMed, Scopus and Web of Science. They screened the title and abstract to retrieve and map out relevant studies on AHLs in oral health, in order to identify key concepts, gaps in knowledge, and areas for further research. This study identified 127 articles and included 42 articles. These studies identified AHLs in human oral samples like saliva, dental plaque, tongue swabs, and dentin caries. The studies also found that AHLs regulate cell-to-cell communication of bacteria (quorum sensing) in mature biofilm fostering the production of virulence factors that damage the immune system. AHLs also exert biological effects on human cells and influence oral diseases such as periodontitis and oral squamous carcinoma. Researchers developed AHL inhibitors to interfere with the quorum sensing process and interrupt the communication between bacteria. These inhibitors can be classified into three main categories based on their mechanisms of action to AHLs: AHL synthesis disruptors, AHL competitive inhibitors and AHL enzymatic degraders. These AHL inhibitors can be important tools in the fight against bacterial infections, particularly those caused by Gram-negative bacteria. The literatures indicate that AHLs, as quorum sensing molecules, influence bacterial communication. AHLs have a significant impact in bacterial pathogencity and play a potential role in the pathogenesis of oral diseases. Researchers have developed AHL inhibitors to disrupt bacterial quorum sensing, preventing bacteria from forming biofilms or expressing virulence factors. These studies on AHLs represent a new research direction to develop novel therapeutic strategies to manage oral diseases.

Albumin orchestrates a natural host defense mechanism against mucormycosis.

Mucormycosis is an emerging, life-threatening human infection caused by fungi of the order Mucorales. Metabolic disorders uniquely predispose an ever-expanding group of patients to mucormycosis via poorly understood mechanisms. Therefore, it is highly likely that uncharacterized host metabolic effectors confer protective immunity against mucormycosis. Here, we uncover a master regulatory role of albumin in host defense against Mucorales through the modulation of the fungal pathogenicity program. Our initial studies identified severe hypoalbuminemia as a prominent metabolic abnormality and a biomarker of poor outcome in independent cohorts of mucormycosis patients. Strikingly, we found that purified albumin selectively inhibits Mucorales growth among a range of human pathogens, and albumin-deficient mice display susceptibility specifically to mucormycosis. The antifungal activity of albumin is mediated by the release of bound free fatty acids (FFAs). Importantly, albumin prevents FFA oxidation, which results in loss of their antifungal properties. A high degree of FFA oxidation is found in the sera of patients with mucormycosis. Physiologically, albumin-bound FFAs blocks the expression of the mycotoxin mucoricin and renders Mucorales avirulent in vivo. Overall, we discovered a novel host defense mechanism that directs the pathogen to suppress its growth and the expression of virulence factors in response to unfavorable metabolic cues regulated by albumin. These findings have major implications for the pathogenesis and management of mucormycosis.

Tuning antibacterial efficacy against Pseudomonas aeruginosa by using green AgNPs in chitosan thin films as a plastic alternative

Nanotechnology advancements have facilitated the development of eco-friendly strategies to combat bacterial infections caused by antibiotic-resistant pathogens. This study promotes a green method for the synthesis of silver nanoparticles (AgNPs) utilizing Eucalyptus globulus leaf extracts as an alternative to traditional colloidal AgNPs obtained through chemical synthesis, investigating their antibacterial efficacy against Pseudomonas aeruginosa and their impact on the expression of bacterial virulence factors (pyocyanin, pyoverdine, rhamnolipids). This work demonstrates that: i. while colloidal AgNPs showed ineffective up to 120 μM, green AgNPs had a bactericidal effect already at 20 μM, without impacting bacterial virulence factors at sub-inhibitory concentrations; ii. the polyphenolic shell surrounding green AgNPs could play a crucial role in the antibacterial mechanisms, with a pro-oxidant action confirmed by a greater sensitivity to hydrogen peroxide (H2O2); iii. AgNPs improved the antibacterial properties of chitosan when incorporated into thin films. Consequently, an environmentally friendly nanocomposite film with antibacterial and antibiofilm properties was produced, which holds promise for application in food packaging to mitigate the emergence of microbial contamination in food products.

Identification, Antimicrobial Resistance Profiling and Virulence Factors of Bacterial Isolates Recovered from Human Clinical Cases

Antimicrobial Resistance (AMR) among different microorganisms has become a serious issue and contributing to cause severe diseases. Proper monitoring measures should be taken to improve the current scenario of antimicrobial resistance. The present study focuses on identification, antimicrobial resistance profiling and virulence factors of bacterial isolates recovered from various human clinical cases. Total 350 clinical samples were collected from a public hospital in Shimla (Himachal Pradesh) and a private diagnostic centre in Mohali (Punjab). K. pneumoniae (51.42%) and E. coli (44.57%) were recovered from all the sample sources while S. aureus (3.15%) and Bacillus spp. (0.86%) were isolated from urine samples only. Overall maximum resistance was observed against ampicillin (94.28%), amoxicillin + clavulanic acid (90.5%), cefepime (88%) and ceftriaxone (80.18%) while it was minimum for cefoperazone (0.85%), kanamycin (1.14%), lomefloxacin and norfloxacin (1.42% each) and cefixime (1.71%). 52.28% isolates were multidrug-resistant (MDR) and 13.42% were extensive drug resistant (XDR). Drug resistant phenotypes were prominently observed in isolates recovered from tracheal fluid and cerebrospinal fluid (CSF). The expression of selective virulence factors like motility, lipase, protease and capsule production was evenly associated with drug-resistant and drug-sensitive bacterial phenotypes; however, siderophore and biofilm production was only seen among isolates with drug resistant phenotype. A significant relation between both the variables was statistically confirmed using chi-square test and the probability value (p &lt; 0.05) for calculated c2 of 43.28 with degree of freedom 10. Occurrence of MDR and XDR bacterial strains among clinical samples bring on economic burden on health system as well as on patient in terms of longer hospital stays and treatment delays.

SarZ inhibits the hemolytic activity through regulation of phenol soluble modulins in Staphylococcus epidermidis.

Staphylococcus epidermidis is an important conditionally pathogenic bacterium. SarZ, belonging to the SarA family protein, has been demonstrated in S. aureus to promote the expression of invasive virulence factors while inhibiting biofilm formation. However, the regulatory role of SarZ on S. epidermidis virulence is not completely understood. In this study, we successfully deleted the sarZ gene by allelic replacement in S. epidermidis. The sarZ mutant strain exhibited remarkably increased hemolytic activity and drastically impaired biofilm formation, suggesting that SarZ is key regulator of virulence in S. epidermidis. Through butanol extraction of the spent medium and HPLC-MS/MS analysis, production of phenol soluble modulins (PSMs) possessing cytolytic effect was found to be elevated significantly in the mutant. Subsequent qRT-PCR experiments demonstrated that expression of the psm genes, especially the β-type, was upregulated dramatically in the mutant. Meanwhile, transcription of icaA gene responsible for biofilm formation was sharply diminished. The sarZ psmβ double mutant was further generated and displayed a significantly decreased hemolytic activity compared with the sarZ mutant. EMSA assays implied that recombinant SarZ protein can directly bind to the promoter regions of the psmβ and ica operon. DNase I footprinting assays further pinpointed two SarZ-binding sites on the psmβ operon promoter. Taken together, the results confirmed that SarZ is a pivotal regulator of virulence in S. epidermidis and might respectively regulate the hemolytic activity and biofilm formation mainly by directly controlling the transcription of psm genes, particularly the β-type, and the ica operon.

Andrographolide protects against the intestinal barrier dysfunction and inflammatory response through modulating ETEC virulence factors in a mouse model of diarrhea

Infectious diarrhea is a common disease that endanger both human and livestock animals. However, there are limited safety strategy for preventing Enterotoxigenic Escherichia coli (ETEC) induced diarrhea. A mouse diarrhea model was established by orally administering ETEC following andrographolide pretreatment. Pretreatment with andrographolide improved the body weight losses, diarrhea, intestinal barrier dysfunction and inflammation of ETEC infected mice. Andrographolide pretreatment protected against the ETEC-induced downregulation of intestinal Na+/H+ exchange protein 3 (Nhe3) and upregulation of cystic fibrosis transmembrane conductance regulator (Cftr) mRNA expression. Andrographolide pretreatment also reduced coliform colonization in the jejunum but did not affect fecal coliform levels in infected mice. By using an in vitro model, it was shown that andrographolide downregulated the expression of virulence factors involved in ETEC k88 colonization, as well as heat-labile and heat-stable enterotoxins. These results unveil a novel finding that andrographolide protect against ETEC infection by regulating its virulence factors.

A Comprehensive Review on the Antibacterial, Antifungal, Antiviral, and Antiparasitic Potential of Silybin.

Silybin, a flavonolignan extracted from the seeds of the plant species Silybum marianum (L.) Gaertn., has a variety of pharmacological activities, including antimicrobial activity against several microorganisms of clinical interest. This review analyzes the existing studies on silybin's antimicrobial activity and possible mechanisms of action. Silybin has been shown to inhibit the growth of Gram-positive and Gram-negative bacteria, as well as some fungi, viruses, and protozoa. In general, possible mechanisms of antimicrobial action include the inhibition of efflux pumps, prevention of biofilm formation, reduction of the expression of virulence factors, induction of apoptosis-like effects, and plasma membrane damage, as well as the inhibition of nucleic acid and protein synthesis. Silybin has been shown to have synergistic effects when combined with conventional antibiotics against both drug-sensitive and drug-resistant microorganisms. However, the low bioavailability observed for this flavonolignan has been a challenge to its clinical use. In this context, nanotechnology has been used to increase silybin's bioavailability while enhancing its antimicrobial activity. Furthermore, certain structural modifications have been able to enhance its antimicrobial activity in comparison to that of the natural molecule. Overall, this review provides insights into the scientific understanding of the mechanism of action of silybin and its desired properties for the effective treatment of infections.