Analyzing the safety of the parasiticide fungus Mucor circinelloides: first insights on its virulence profile and interactions with the avian gut microbial community.

Parasiticide fungi are considered an accurate, sustainable, and safe solution for the biocontrol of animal gastrointestinal (GI) parasites. This research provides an initial characterization of the virulence of the native parasiticide fungus Mucor circinelloides (FMV-FR1) and an assessment of its impact on birds' gut microbes. The genome of this fungus was sequenced to identify the genes coding for virulence factors. Also, this fungus was checked for the phenotypic expression of proteinase, lecithinase, DNase, gelatinase, hemolysin, and biofilm production. Finally, an in vivo trial was developed based on feeding M. circinelloides spores to laying hens and peacocks three times a week. Bird feces were collected for 3 months, with total genomic DNA being extracted and subjected to long-read 16S and 25S-28S sequencing. Genes coding for an iron permease (FTR1), iron receptors (FOB1 and FOB2), ADP-ribosylation factors (ARFs) (ARF2 and ARF6), and a GTPase (CDC42) were identified in this M. circinelloides genome. Also, this fungus was positive only for lecithinase activity. The field trial revealed a fecal microbiome dominated by Firmicutes and Proteobacteria in laying hens, and Firmicutes and Bacteroidetes in peacocks, whereas the fecal mycobiome of both bird species was mainly composed of Ascomycetes and Basidiomycetes fungi. Bacterial and fungal alpha-diversities did not differ between sampling time points after M. circinelloides administrations (P = 0.62 and P = 0.15, respectively). Although findings from this research suggest the lack of virulence of this M. circinelloides parasiticide isolate, more complementary in vitro and in vivo research is needed to conclude about the safety of its administration to birds, aiming at controlling their GI parasites.IMPORTANCEA previous study revealed that the native Mucor circinelloides isolate (FMV-FR1) can develop parasiticide activity toward coccidia oocysts, one of the most pathogenic GI parasites in birds. However, ensuring its safety for birds is of utmost importance, namely by studying its virulence profile and potential effect on commensal gut microbes. This initial study revealed that although this M. circinelloides isolate had genes coding for four types of virulence factors-iron permease, iron receptors, ADP-ribosylation factors, and GTPase-and only expressed phenotypically the enzyme lecithinase, the administration of its spores to laying hens and peacocks did not interfere with the abundances and diversities of their gut commensal bacteria and fungi. Although overall results suggest the lack of virulence of this M. circinelloides isolate, more complementary research is needed to conclude about the safety of its administration to birds in the scope of parasite biocontrol programs.

Effect of Polyphenols on Inflammation Induced by Membrane Vesicles from Staphylococcus aureus.

Staphylococcus aureus, a bacterium found on human skin, produces toxins and various virulence factors that can lead to skin infections such as atopic dermatitis. These toxins and virulence factors are carried in membrane vesicles (MVs), composed of the bacterium's own cell membranes, and are expected to reach host target cells in a concentrated form, inducing inflammation. This study investigated the effects of two polyphenols, (-)-epigallocatechin gallate (EGCG) and nobiletin (NOL), on the expression of S. aureus virulence factors and the inflammation induced by MVs. The study found that EGCG alone decreased the production of Staphylococcal Enterotoxin A (SEA), while both EGCG and NOL reduced biofilm formation and the expression of virulence factor-related genes. When S. aureus was cultured in a broth supplemented with these polyphenols, the resulting MVs showed a reduction in SEA content and several cargo proteins. These MVs also exhibited decreased levels of inflammation-related gene expression in immortalized human keratinocytes. These results suggest that EGCG and NOL are expected to inhibit inflammation in the skin by altering the properties of MVs derived from S. aureus.

Mechanistic research: Selenium regulates virulence factors, reducing adhesion ability and inflammatory damage of Helicobacter pylori.

BACKGROUND: The pathogenicity of Helicobacter pylori is dependent on factors including the environment and the host. Although selenium is closely related to pathogenicity as an environmental factor, the specific correlation between them remains unclear. AIM: To investigate how selenium acts on virulence factors and reduces their toxicity. METHODS: H. pylori strains were induced by sodium selenite. The expression of cytotoxin-associated protein A (CagA) and vacuolating cytotoxin gene A (VacA) was determined by quantitative PCR and Western blotting. Transcriptomics was used to analyze CagA, CagM, CagE, Cag1, Cag3, and CagT. C57BL/6A mice were infected with the attenuated strains subjected to sodium selenite induction, and H. pylori colonization, inflammatory reactions, and the cell adhesion ability of H. pylori were assessed. RESULTS: CagA and VacA expression was upregulated at first and then downregulated in the H. pylori strains after sodium selenite treatment. Their expression was significantly and steadily downregulated after the 5th cycle (10 d). Transcriptome analysis revealed that sodium selenite altered the levels affect H. pylori virulence factors such as CagA, CagM, CagE, Cag1, Cag3, and CagT. Of these factors, CagM and CagE expression was continuously downregulated and further downregulated after 2 h of induction with sodium selenite. Moreover, CagT expression was upregulated before the 3rd cycle (6 d) and significantly downregulated after the 5th cycle. Cag1 and Cag3 expression was upregulated and downregulated, respectively, but no significant change was observed by the 5th cycle. C57BL/6A mice were infected with the attenuated strains subjected to sodium selenite induction. The extent of H. pylori colonization in the stomach increased; however, sodium selenite also induced a mild inflammatory reaction in the gastric mucosa of H. pylori-infected mice, and the cell adhesion ability of H. pylori was significantly weakened. CONCLUSION: These results demonstrate that H. pylori displayed virulence attenuation after the 10th d of sodium selenite treatment. Sodium selenite is a low toxicity compound with strong stability that can reduce the cell adhesion ability of H. pylori, thus mitigating the inflammatory damage to the gastric mucosa.

Antimicrobial and anti-virulence activities of 4-shogaol from grains of paradise against gram-negative bacteria: Integration of experimental and computational methods.

ETHNOPHARMACOLOGICAL RELEVANCE: Bacterial resistance to antibiotics is a growing global concern, highlighting the urgent need for new antimicrobial candidates. Aframomum melegueta was traditionally used for combating urinary tract and soft tissue infections, which implies its potential as an antimicrobial agent. AIM OF STUDY: This study was designed to explore the antibacterial and anti-virulence capabilities of 4-shogaol isolated from A. melegueta seeds versus gram-negative bacteria: Serratia marcescens, Klebsiella pneumoniae, Acinetobacter baumannii, and the clinically important pathogen Pseudomonas aeruginosa. MATERIALS AND METHODS: 4-Shogeol was isolated from A. melegueta seeds and its MICs were determined for Acinetobacter baumannii (ATCC-17978), Pseudomonas aeruginosa (ATCC-27853), Klebsiella pneumoniae (ATCC-700603), and Serratia marcescens clinical isolate. The anti-efflux activity and effect on the bacterial cell membrane for the compound were evaluated. Furthermore, the anti-virulence activities of the compound were evaluated. The effects of 4-shogeol at sub-MIC on bacterial motility, biofilm formation, and production of virulent enzymes and pigments were assessed. The anti-quorum sensing activities of 4-shogeol were evaluated virtually and by quantification its effect on the expression of quorum sensing encoding genes. The in vivo protection assay was conducted to evaluate the effect of 4-shogaol on the P. aeruginosa capacity to induce pathogenesis in mice. Finally, the effect of shogaol-antibiotics combination was assessed. RESULTS: The research revealed that 4-shogaol's antibacterial action primarily involves disrupting the bacterial cell membrane and efflux pumps. It also exhibited significant anti-virulence effects by reducing biofilm development and repressing virulence factors production, effectively protecting mice against P. aeruginosa infection. Furthermore, when combined with antibiotics, 4-shogaol demonstrated synergistic effects, leading to reduced minimum inhibitory concentrations (MICs) against P. aeruginosa. Its anti-virulence properties were linked to its ability to disrupt bacterial quorum sensing (QS) mechanisms, as evidenced by its interaction with QS receptors and downregulation of QS-related genes. Notably, in silico analysis indicated that 4-shogaol exhibited strong binding affinity to different P. aeruginosa QS targets. CONCLUSION: These findings suggest that 4-shogaol holds promise as an effective anti-virulence agent that can be utilized in combination with antibiotics for treating severe infections caused by gram-positive bacteria.

Antibiofilm and anti-quorum sensing activity of Psidium guajava L. leaf extract: In vitro and in silico approach.

The quorum sensing mechanism relies on the detection and response to chemical signals, termed autoinducers, which regulate the synthesis of virulence factors including toxins, enzymes, and biofilms. Emerging therapeutic strategies for infection control encompass approaches that attenuate quorum-sensing systems. In this study, we evaluated the antibacterial, anti-quorum sensing, and anti-biofilm activities of Psidium guajava L. methanolic leaf extracts (PGME). Minimum Inhibitory Concentrations (MICs) of PGME were determined as 500 µg/ml for C. violaceum and 1000 µg/ml for P. aeruginosa PAO1. Significantly, even at sub-MIC concentrations, PGME exhibited noteworthy anti-quorum sensing properties, as evidenced by concentration-dependent inhibition of pigment production in C. violaceum 12742. Furthermore, PGME effectively suppressed quorum-sensing controlled virulence factors in P. aeruginosa PAO1, including biofilm formation, pyoverdin, pyocyanin, and rhamnolipid production, with concentration-dependent inhibitory effects. Phytochemical analysis utilizing GC-MS revealed the presence of compounds such as alpha-copaene, caryophyllene, and nerolidol. In-silico docking studies indicated a plausible mechanism for the observed anti-quorum sensing activity, involving favorable binding and interactions with QS-receptors, including RhlR, CviR', LasI, and LasR proteins. These interactions were found to potentially disrupt QS pathways through suppression of AHL production and receptor protein blockade. Collectively, our findings propose PGME as a promising candidate for the treatment of bacterial infections. Its attributes that mitigate biofilm development and impede quorum-sensing mechanisms highlight its potential therapeutic value.

A bacterial pathogen induces developmental slowing by high reactive oxygen species and mitochondrial dysfunction in Caenorhabditis elegans.

Host-pathogen interactions are complex by nature, and the host developmental stage increases this complexity. By utilizing Caenorhabditis elegans larvae as the host and the bacterium Pseudomonas aeruginosa as the pathogen, we investigated how a developing organism copes with pathogenic stress. By screening 36 P. aeruginosa isolates, we found that the CF18 strain causes a severe but reversible developmental delay via induction of reactive oxygen species (ROS) and mitochondrial dysfunction. While the larvae upregulate mitophagy, antimicrobial, and detoxification genes, mitochondrial unfolded protein response (UPRmt) genes are repressed. Either antioxidant or iron supplementation rescues the phenotypes. We examined the virulence factors of CF18 via transposon mutagenesis and RNA sequencing (RNA-seq). We found that non-phenazine toxins that are regulated by quorum sensing (QS) and the GacA/S system are responsible for developmental slowing. This study highlights the importance of ROS levels and mitochondrial health as determinants of developmental rate and how pathogens can attack these important features.

Genomes of four Streptomyces strains reveal insights into putative new species and pathogenicity of scab-causing organisms.

Genomes of four Streptomyces isolates, two putative new species (Streptomyces sp. JH14 and Streptomyces sp. JH34) and two non thaxtomin-producing pathogens (Streptomyces sp. JH002 and Streptomyces sp. JH010) isolated from potato fields in Colombia were selected to investigate their taxonomic classification, their pathogenicity, and the production of unique secondary metabolites of Streptomycetes inhabiting potato crops in this region. The average nucleotide identity (ANI) value calculated between Streptomyces sp. JH34 and its closest relatives (92.23%) classified this isolate as a new species. However, Streptomyces sp. JH14 could not be classified as a new species due to the lack of genomic data of closely related strains. Phylogenetic analysis based on 231 single-copy core genes, confirmed that the two pathogenic isolates (Streptomyces sp. JH010 and JH002) belong to Streptomyces pratensis and Streptomyces xiamenensis, respectively, are distant from the most well-known pathogenic species, and belong to two different lineages. We did not find orthogroups of protein-coding genes characteristic of scab-causing Streptomycetes shared by all known pathogenic species. Most genes involved in biosynthesis of known virulence factors are not present in the scab-causing isolates (Streptomyces sp. JH002 and Streptomyces sp. JH010). However, Tat-system substrates likely involved in pathogenicity in Streptomyces sp. JH002 and Streptomyces sp. JH010 were identified. Lastly, the presence of a putative mono-ADP-ribosyl transferase, homologous to the virulence factor scabin, was confirmed in Streptomyces sp. JH002. The described pathogenic isolates likely produce virulence factors uncommon in Streptomyces species, including a histidine phosphatase and a metalloprotease potentially produced by Streptomyces sp. JH002, and a pectinesterase, potentially produced by Streptomyces sp. JH010. Biosynthetic gene clusters (BGCs) showed the presence of clusters associated with the synthesis of medicinal compounds and BGCs potentially linked to pathogenicity in Streptomyces sp. JH010 and JH002. Interestingly, BGCs that have not been previously reported were also found. Our findings suggest that the four isolates produce novel secondary metabolites and metabolites with medicinal properties.

The Application of Cinnamon Twig Extract as an Inhibitor of Listeriolysin O against Listeria monocytogenes Infection.

As a major virulence factor of Listeria monocytogenes (L. monocytogenes), listeriolysin O (LLO) can assist in the immune escape of L. monocytogenes, which is critical for the pathogen to evade host immune recognition, leading to various infectious diseases. Cinnamon twig (CT), as a traditional medicine, has been widely used in clinics for multiple functions and it has exhibited excellent safety, efficacy and stability. There are few reports on the effects of the extracts of traditional medicine on bacterial virulence factors. CT has not been reported to be effective in the treatment of L. monocytogenes infection. Therefore, this study aims to explore the preventive effect of CT against L. monocytogenes infection in vivo and in vitro by targeting LLO. Firstly, a hemolysis assay and a cell viability determination are used to detect the effect of CT extract on the inhibition of the cytolytic activity of LLO. The potential mechanism through which CT extract inhibits LLO activity is predicted through network pharmacology, molecular docking assay, real-time polymerase chain reaction (RT-PCR), Western blotting and circular dichroism (CD) analysis. The experimental therapeutic effect of CT extract is examined in a mouse model infected with L. monocytogenes. Then, the ingredients are identified through a high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) analysis. Here we find that CT extract, containing mainly cinnamic acid, cinnamaldehyde, ß-sitosterol, taxifolin, catechin and epicatechin, shows a potential inhibition of LLO-mediated hemolysis without any antimicrobial activity. The results of the mechanism research show that CT extract treatment can simultaneously inhibit LLO expression and oligomerization. Furthermore, the addition of CT extract led to a remarkable alleviation of LLO-induced cytotoxicity. After treatment with CT extract, the mortality, bacterial load, pathological damage and inflammatory responses of infected mice are significantly reduced when compared with the untreated group. This study suggests that CT extract can be a novel and multicomponent inhibitor of LLO with multiple strategies against L. monocytogenes infection, which could be further developed into a novel treatment for infections caused by L. monocytogenes.

Genome-scale model of Pseudomonas aeruginosa metabolism unveils virulence and drug potentiation.

Pseudomonas aeruginosa is one of the leading causes of hospital-acquired infections. To decipher the metabolic mechanisms associated with virulence and antibiotic resistance, we have developed an updated genome-scale model (GEM) of P. aeruginosa. The model (iSD1509) is an extensively curated, three-compartment, and mass-and-charge balanced BiGG model containing 1509 genes, the largest gene content for any P. aeruginosa GEM to date. It is the most accurate with prediction accuracies as high as 92.4% (gene essentiality) and 93.5% (substrate utilization). In iSD1509, we newly added a recently discovered pathway for ubiquinone-9 biosynthesis which is required for anaerobic growth. We used a modified iSD1509 to demonstrate the role of virulence factor (phenazines) in the pathogen survival within biofilm/oxygen-limited condition. Further, the model can mechanistically explain the overproduction of a drug susceptibility biomarker in the P. aeruginosa mutants. Finally, we use iSD1509 to demonstrate the drug potentiation by metabolite supplementation, and elucidate the mechanisms behind the phenotype, which agree with experimental results.

Virulence Induction in Pseudomonas aeruginosa under Inorganic Phosphate Limitation: a Proteomics Perspective.

Inorganic phosphate (Pi) is a central nutrient and signal molecule for bacteria. Pi limitation was shown to increase the virulence of several phylogenetically diverse pathogenic bacteria with different lifestyles. Hypophosphatemia enhances the risk of death in patients due to general bacteremia and was observed after surgical injury in humans. Phosphate therapy, or the reduction of bacterial virulence by the administration of Pi or phosphate-containing compounds, is a promising anti-infective therapy approach that will not cause cytotoxicity or the emergence of antibiotic-resistant strains. The proof of concept of phosphate therapy has been obtained using primarily Pseudomonas aeruginosa (PA). However, a detailed understanding of Pi-induced changes at protein levels is missing. Using pyocyanin production as proxy, we show that the Pi-mediated induction of virulence is a highly cooperative process that occurs between 0.2 to 0.6 mM Pi. We present a proteomics study of PA grown in minimal medium supplemented with either 0.2 mM or 1 mM Pi and rich medium. About half of the predicted PA proteins could be quantified. Among the 1,471 dysregulated proteins comparing growth in 0.2 mM to 1 mM Pi, 1,100 were depleted under Pi-deficient conditions. Most of these proteins are involved in general and energy metabolism, different biosynthetic and catabolic routes, or transport. Pi depletion caused accumulation of proteins that belong to all major families of virulence factors, including pyocyanin synthesis, secretion systems, quorum sensing, chemosensory signaling, and the secretion of proteases, phospholipases, and phosphatases, which correlated with an increase in exoenzyme production and antibacterial activity. IMPORTANCE Antibiotics are our main weapons to fight pathogenic bacteria, but the increase in antibiotic-resistant strains and their consequences represents a major global health challenge, revealing the necessity to develop alternative antimicrobial strategies that do not involve the bacterial killing or growth inhibition. P. aeruginosa has been placed second on the global priority list to guide research on the development of new antibiotics. One of the most promising alternative strategies is the phosphate therapy for which the proof of concept has been obtained for P. aeruginosa. This article reports the detailed changes at the protein levels comparing P. aeruginosa grown under Pi-abundant and Pi-depleted conditions. These data describe in detail the molecular mechanisms underlying phosphate therapy. Apart from Pi, several other phosphate-containing compounds have been used for phosphate therapy and this study will serve as a reference for comparative studies aimed at evaluating the effect of alternative compounds.

Acinetobacter baumannii: Pathogenesis, virulence factors, novel therapeutic options and mechanisms of resistance to antimicrobial agents with emphasis on tigecycline.

WHAT IS KNOWN AND OBJECTIVE: Acinetobacter baumannii is one of the most important nosocomial pathogens with the ability to cause infections such as meningitis, pneumonia, urinary tract, septicaemia and wound infections. A wide range of virulence factors are responsible for pathogenesis and high mortality of A. baumannii including outer membrane proteins, lipopolysaccharide, capsule, phospholipase, nutrient- acquisition systems, efflux pumps, protein secretion systems, quarom sensing and biofilm production. These virulence factors contribute in pathogen survival in stressful conditions and antimicrobial resistance. COMMENT: According to the World Health Organization (WHO), A. baumannii is one of the most resistant pathogens of ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa and Enterobacter spp.). In recent years, resistance to a wide range of antibiotics in A. baumannii has significantly increased and the high emergence of extensively drug resistant (XDR) isolates is challenging. Among therapeutic antibiotics, resistance to tigecycline as a last resort antibiotic has become a global concern. Several mechanisms are involved in tigecycline resistance, the most important of which is RND (Resistance-Nodulation-Division) family efflux pumps overexpression. The development of new therapeutic strategies to confront A. baumannii infections has been very promising in recent years. WHAT IS NEW AND CONCLUSION: In the present review we highlight microbiological and virulence traits in A. baumannii and peruse the tigecycline resistance mechanisms and novel therapeutic options. Among the novel therapeutic strategies we focus on combination therapy, drug repurposing, novel antibiotics, bacteriophage therapy, antimicrobial peptides (AMPs), human monoclonal antibodies (Hu-mAbs), nanoparticles and gene editing.

20S-ginsenoside Rg3 inhibits the biofilm formation and haemolytic activity of Staphylococcus aureus by inhibiting the SaeR/SaeS two-component system.

Introduction. Staphylococcus aureus is a major cause of chronic diseases and biofilm formation is a contributing factor. 20S-ginsenoside Rg3 (Rg3) is a natural product extracted from the traditional Chinese medicine red ginseng.Gap statement. The effects of Rg3 on biofilm formation and haemolytic activity as well as its antibacterial mechanism against S. aureus have not been reported.Aim. This study aimed to investigate the effects of Rg3 on biofilm formation and haemolytic activity as well as its antibacterial action against clinical S. aureus isolates.Methodology. The effect of Rg3 on biofilm formation of clinical S. aureus isolates was studied by crystal violet staining. Haemolytic activity analysis was carried out. Furthermore, the influence of Rg3 on the proteome profile of S. aureus was studied by quantitative proteomics to clarify the mechanism underlying its antibacterial action and further verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).Results. Rg3 significantly inhibited biofilm formation and haemolytic activity in clinical S. aureus isolates. A total of 63 with >1.5-fold changes in expression were identified, including 34 upregulated proteins and 29 downregulated proteins. Based on bioinformatics analysis, the expression of several virulence factors and biofilm-related proteins, containing CopZ, CspA, SasG, SaeR/SaeS two-component system and SaeR/SaeS-regulated proteins, including leukocidin-like protein 2, immunoglobulin-binding protein G (Sbi) and fibrinogen-binding protein, in the S. aureus of the Rg3-treated group was downregulated. RT-qPCR confirmed that Rg3 inhibited the regulation of SaeR/SaeS and decreased the transcriptional levels of the biofilm-related genes CopZ, CspA and SasG.Conclusions. Rg3 reduces the formation of biofilm by reducing cell adhesion and aggregation. Further, Rg3 can inhibit the SaeR/SaeS two-component system, which acts as a crucial signal transduction system for the anti-virulence activity of Rg3 against clinical S. aureus isolates.

Anti-quorum sensing, antibiofilm, and antibacterial activities of extracts of Centella asiatica L. leaves, and in vitro derived leaves-calli through tissue culture: a potential for biofouling-prevention.

Extracts of Centella asiatica leaves (LEs), and in-vitro leaf-calli (CEs), were investigated for antibacterial, antibiofilm, and anti-quorum sensing activities. Ethyl acetate extracts from leaves (EALE), leaf-calli (EACE), methanolic extracts from leaves (MELE), and leaf-calli (MECE) showed antibacterial activity; the minimum inhibitory concentrations (MICs) of LEs and CEs ranged from 0.312-2.50 mg ml-1 and 0.625 - 2.50 mg ml-1, respectively. The MICs of EALE and EACE were 2.50 mg ml-1, each, for C. violaceum 12742, and P. aeruginosa PAO1. At sub-MIC levels, EALE and EACE showed anti-quorum sensing (anti-QS) activity, demonstrated by concentration dependent pigment inhibition of C. violaceum 12742. Similarly, EALE and EACE inhibited QS-controlled virulence factors in P. aeruginosa PAO1 (biofilm, pyocyanin, and pyoverdin); again, the inhibition was concentration-dependent. The best effect was at immediate sub-MIC concentration i.e. 1250 µg ml-1. GC-MS analyses revealed the presence of compound 9,12-Octadecadienoic acid, and in silico docking study suggested interactions with QS-receptors CviR', LasI, and LasR proteins for anti-QS activity.

Molecular investigation of prevalence, phenotypic and genotypic diversity, antibiotic resistance, frequency of virulence genes and genome sequencing in Pseudomonas aeruginosa strains isolated from lobster.

AIM: Aquatic organisms are too susceptible to the increased growth of bacterial contamination. It seems that preventive measures should be prioritized to reduce bacterial load, and improve the health situation of marine-based product consumers. Hence, this study is aimed at molecular investigation of the prevalence of Pseudomonas aeruginosa as one of the most food-borne pathogens, antibiotic resistance, and virulence factor encoding gens in lobster samples. METHODOLOGY: After the collection of aquatic samples from Isfahan and Chabahar city during the summer and autumn seasons, they were cultured, and confirmed by biochemistry tests. Then, they were investigated for antibiotic resistance by the Kirby Bauer method. Then, antibiotic resistance, virulence factor encoding genes, and Multi-Drug Resistance (MDR) patterns were analyzed. Statistical analysis was done by SPSS through chi-square tests. RESULTS: Bacterial contamination in samples taken from Isfahan city was higher than in Chabahar city despite having a cooler climate on summer days. Antibiotic resistance to piperacillin in fresh shrimp samples taken in summer In Isfahan city was contrary to its usage as a front-line antibiotic agent for Pseudomonas aeruginosa. Lowered MDR pattern in frozen samples, was related to the varied expression of antibiotic resistance, highlighting the importance of regulations for cold chain in storage, transportation, and distribution of marine samples, especially when compared to fresh shrimps. CONCLUSION: Food-borne pathogens, antibiotic resistance, and their virulence factors are of clinical and environmental importance. Results of our study indicated a high rate of frequency for Pseudomonas aeruginosa isolated from marine samples, antibiotic resistance, antibiotic resistance encoding genes, virulence factors encoding genes, and MDR. Maintenance of the cold chain, and proper food processing, have indispensable roles in the preservation, and reduction of Pseudomonas aeruginosa frequency in aquatic organisms.

Photo-oxidative stress response and virulence traits are co-regulated in E. faecalis after antimicrobial photodynamic therapy.

Knowledge of photo-oxidative stress responses in bacteria that survive antimicrobial photodynamic therapy (aPDT) is scarce. Whereas aPDT is attracting growing clinical interest, subsequent stress responses are crucial to evaluate as they may lead to the up-regulation of pathogenic traits. Here, we aimed to assess transcriptional responses to sublethal aPDT-stress and identify potential connections with virulence-related genes. Six Enterococcus faecalis strains were investigated; ATCC 29212, three dental root-canal isolates labelled UmID1, UmID2 and UmID3 and two vancomycin-resistant isolates labelled A1 and A2. TMPyP was employed as a photosensitiser. A viability dose-response curve to increasing concentrations of TMPyP was determined by culture plating. Differential expression of genes involved in oxidative stress responses (dps and hypR), general stress responses (dnaK, sigma-factorV and relA), virulence-related genes (ace, fsrC and gelE) and vancomycin-resistance (vanA) was assessed by reverse-transcription qPCR. TMPyP-mediated aPDT inactivated all strains with comparable efficiencies. TMPyP at 0.015 µM was selected to induce sublethal photo-oxidative stress. Despite heterogeneities in gene expression between strains, transcriptional profiles revealed up-regulations of transcripts dps, hypR as well as dnaK and sigma factorV after exposure to TMPyP alone and to light-irradiated TMPyP. Specifically, the alternative sigma factorV reached up to 39 ± 113-fold (median ± IQR) (p = 0.0369) in strain A2. Up-regulation of the quorum sensing operon, fsr, and its downstream virulence-related gelatinase gelE were also observed in strains ATCC-29212, A1, A2 and UmID3. Finally, photo-oxidative stress induced vanA-type vancomycin-resistance gene in both carrier isolates, reaching up to 3.3 ± 17-fold in strain A2 (p = 0.015). These findings indicate that, while aPDT successfully inactivates vancomycin-resistant and naïve strains of E. faecalis, subpopulations of surviving cells respond by co-ordinately up-regulating a network of genes involved in stress survival and virulence. This includes the induction of vancomycin-resistance genes in carrier isolates. These data may provide the mechanistic basis to circumvent bacterial responses and improve future clinical protocols.

Effects of the green tea catechin epigallocatechin-3-gallate on Streptococcus mutans planktonic cultures and biofilms: systematic literature review of in vitro studies.

This study aimed at performing a systematic review of the literature on the effects of epigallocatechin-3-gallate (EGCG) on Streptococcus mutans planktonic cultures and biofilms. The selected references demonstrated that EGCG suppresses S. mutans acid production by inhibiting the activity of enzymes such as lactate dehydrogenase and FIF0-ATPase. Regarding virulence factors, one study reported a reduction in soluble and insoluble polysaccharide synthesis, another demonstrated that EGCG inhibited GTase activity, and another showed effects of EGCG on the expression of gtf B, C, and D. The effects of EGCG on S. mutans biofilms were reported only by 2 of the selected studies. Moreover, high variability in effective concentrations and microbial assessment methods were observed. The literature suggests that EGCG has effects against S. mutans planktonic cells viability and virulence factors. However, the literature lacks studies with appropriate biofilm models to evaluate the precise effectiveness of EGCG against S. mutans biofilms.

Therapeutic Inhibition of Staphylococcus aureus ArlRS Two-Component Regulatory System Blocks Virulence.

Staphylococcus aureus is a common cause of severe infections, and its widespread antibiotic resistance necessitates search for alternative therapies, such as inhibition of virulence. As S. aureus produces multiple individual virulence factors, inhibition of an entire regulatory system might provide better effects than targeting each virulence factor separately. Herein, we describe two novel inhibitors of S. aureus two-component regulatory system ArlRS: 3,4'-dimethoxyflavone and homopterocarpin. Unlike other putative ArlRS inhibitors previously identified, these two compounds were effective and specific. In vitro kinase assays indicated that 3,4'-dimethoxyflavone directly inhibits ArlS autophosphorylation, while homopterocarpin did not exhibit such effect, suggesting that two inhibitors work through distinct mechanisms. Application of the inhibitors to methicillin-resistant S. aureus (MRSA) in vitro blocked ArlRS signaling, inducing an abnormal gene expression pattern that was reflected in changes at the protein level, enhanced sensitivity to oxacillin, and led to the loss of numerous cellular virulence traits, including the ability to clump, adhere to host ligands, and evade innate immunity. The pleiotropic antivirulence effect of inhibiting a single regulatory system resulted in a marked therapeutic potential, demonstrated by the ability of inhibitors to decrease severity of MRSA infection in mice. Altogether, this study demonstrated the feasibility of ArlRS inhibition as anti-S. aureus treatment, and identified new lead compounds for therapeutic development.

(MeOPhSe)2, a synthetic organic selenium compound, inhibits virulence factors of Candida krusei: Adherence to cervical epithelial cells and biofilm formation.

BACKGROUND: Systemic candidiasis is produced by Candida albicans or non-albicans Candida species, opportunistic fungi that produce both superficial and invasive infections. Despite the availability of a wide range of antifungal agents for the treatment of candidiasis, failure of therapy is observed frequently, which opens new avenues in the field of alternative therapeutic strategies. METHODS: The effects of p,p'-methoxyl-diphenyl diselenide [(MeOPhSe)2], a synthetic organic selenium (organochalcogen) compound, were investigated on virulence factors of C. krusei and compared with its antifungal effects on the virulence factors related to adhesion to cervical epithelial cell surfaces with C. albicans. RESULTS: (MeOPhSe)2, a compound non-toxic in epithelial (HeLa) and fibroblastic (Vero) cells, inhibited the growth in a dose-dependent manner and changed the kinetics parameters of C. krusei and, most importantly, extending the duration of lag phase of growth, inhibiting biofilm formation, and changing the structure of biofilm. Also, (MeOPhSe)2 reduced C. albicans and C. krusei adherence to cervical epithelial cells, an important factor for the early stage of the Candida-host interaction. The reduction was 37.24 ± 2.7 % in C. krusei (p = 0.00153) and 32.84 ± 3.2 % in C. albicans (p = 0.0072) at 20 µM (MeOPhSe)2, and the effect is in a concentration-dependent manner. Surprisingly, the antifungal potential on adhesion was similar between both species, indicating the potential of (MeOPhSe)2 as a promising antifungal drug against different Candida infections. CONCLUSION: Overall, we demonstrated the potential of (MeOPhSe)2 as an effective antifungal drug against the virulence factors of Candida species.

Traditional Chinese Medicine Tanreqing Targets Both Cell Division and Virulence in Staphylococcus aureus.

Staphylococcus aureus has been recognized as an important human pathogen and poses a serious health threat worldwide. With the advent of antibiotic resistance, such as the increased number of methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to develop new therapeutical agents. In this study, Chinese traditional medicine Tanreqing (TRQ) has been used as an alternative treating agent against MRSA and we aim to unravel the mode of action of TRQ underlying MRSA inhibition. TRQ treatment affected numerous gene expression as revealed by RNA-seq analysis. Meanwhile, TRQ targeted cell division to inhibit cell growth as shown by illumination microscopy. Besides, we confirmed that TRQ downregulates the expression of virulence factors such as hemolysin and autolysin. Finally, we used a murine model to demonstrate that TRQ efficiently reduces bacterial virulence. Altogether, we have proved TRQ formula to be an effective agent against S. aureus infections.

The Yin and Yang of Pneumolysin During Pneumococcal Infection.

Pneumolysin (PLY) is a pore-forming toxin produced by the human pathobiont Streptococcus pneumoniae, the major cause of pneumonia worldwide. PLY, a key pneumococcal virulence factor, can form transmembrane pores in host cells, disrupting plasma membrane integrity and deregulating cellular homeostasis. At lytic concentrations, PLY causes cell death. At sub-lytic concentrations, PLY triggers host cell survival pathways that cooperate to reseal the damaged plasma membrane and restore cell homeostasis. While PLY is generally considered a pivotal factor promoting S. pneumoniae colonization and survival, it is also a powerful trigger of the innate and adaptive host immune response against bacterial infection. The dichotomy of PLY as both a key bacterial virulence factor and a trigger for host immune modulation allows the toxin to display both "Yin" and "Yang" properties during infection, promoting disease by membrane perforation and activating inflammatory pathways, while also mitigating damage by triggering host cell repair and initiating anti-inflammatory responses. Due to its cytolytic activity and diverse immunomodulatory properties, PLY is integral to every stage of S. pneumoniae pathogenesis and may tip the balance towards either the pathogen or the host depending on the context of infection.