Non-aureus staphylococci and mammaliicocci (NASM): their role in bovine mastitis and One Health.

Non-aureus staphylococci (NAS) are gaining importance in mastitis and public health, and some NAS have been reclassified as mammaliicocci (NASM). Bovine milk production has a major influence on the world economy, being an essential source of income for small, medium and large producers, and bovine mastitis caused by NASM can cause an economic impact. Mastitis generates financial losses due to reduced revenue, increased veterinary costs and expenses associated with animal slaughter. However, it is also a public health issue involving animal health and welfare, human health and the ecosystem. Furthermore, it is an increasingly common infection caused by NASM, including antimicrobial-resistant strains. Despite all these adverse effects that NASM can cause, some studies also point to its protective role against mastitis. Therefore, this review article addresses the negative and positive aspects that NASM can cause in bovine mastitis, the virulence of the disease and resistance factors that make it difficult to treat and, through the One Health approach, presents a holistic view of how mastitis caused by NASM can affect both animal and human health at one and the same time.

ZntA maintains zinc and cadmium homeostasis and promotes oxidative stress resistance and virulence in Vibrio parahaemolyticus.

Although metals are essential for life, they are toxic to bacteria in excessive amounts. Therefore, the maintenance of metal homeostasis is critical for bacterial physiology and pathogenesis. Vibrio parahaemolyticus is a significant food-borne pathogen that mainly causes acute gastroenteritis in humans and acute hepatopancreatic necrosis disease in shrimp. Herein, we report that ZntA functions as a zinc (Zn) and cadmium (Cd) homeostasis mechanism and contributes to oxidative stress resistance and virulence in V. parahaemolyticus. zntA is remarkably induced by Zn, copper, cobalt, nickel (Ni), and Cd, while ZntA promotes V. parahaemolyticus growth under excess Zn/Ni and Cd conditions via maintaining Zn and Cd homeostasis, respectively. The growth of ΔzntA was inhibited under iron (Fe)-restricted conditions, and the inhibition was associated with Zn homeostasis disturbance. Ferrous iron supplementation improved the growth of ΔzntA under excess Zn, Ni or Cd conditions. The resistance of ΔzntA to H2O2-induced oxidative stress also decreased, and its virulence was attenuated in zebrafish models. Quantitative real-time PCR, mutagenesis, and ß-galactosidase activity assays revealed that ZntR positively regulates zntA expression by binding to its promoter. Collectively, the ZntR-regulated ZntA is crucial for Zn and Cd homeostasis and contributes to oxidative stress resistance and virulence in V. parahaemolyticus.

Copper affects virulence and diverse phenotypes of uropathogenic Proteus mirabilis.

BACKGROUND: Copper plays a role in urinary tract infection (UTI) and urinary copper content is increased during Proteus mirabilis UTI. We therefore investigated the effect of copper on uropathogenic P. mirabilis and the underlying mechanisms, focusing on the virulence associated aspects. METHODS: Mouse colonization, swarming/swimming assays, measurement of cell length, flagellin level and urease activity, adhesion/invasion assay, biofilm formation, killing by macrophages, oxidative stress susceptibility, OMPs analysis, determination of MICs and persister cell formation, RT-PCR and transcriptional reporter assay were performed. RESULTS: We found that copper-supplemented mice were more resistant to be colonized in the urinary tract, together with decreased swarming/swimming, ureases activity, expression of type VI secretion system and adhesion/invasion to urothelial cells and increased killing by macrophages of P. mirabilis at a sublethal copper level. However, bacterial biofilm formation and resistance to oxidative stress were enhanced under the same copper level. Of note, the presence of copper led to increased ciprofloxacin MIC and more persister cell formation against ampicillin. In addition, the presence of copper altered the outer membrane protein profile and triggered expression of RcsB response regulator. For the first time, we unveiled the pleiotropic effects of copper on uropathogenic P. mirabilis, especially for induction of bacterial two-component signaling system regulating fitness and virulence. CONCLUSION: The finding of copper-mediated virulence and fitness reinforced the importance of copper for prevention and therapeutic interventions against P. mirabilis infections. As such, this study could facilitate the copper-based strategies against UTI by P. mirabilis.

L-arginine supplementation abrogates hypoxia-induced virulence of Staphylococcus aureus in a murine diabetic pressure wound model.

Diabetic foot ulcers (DFUs) are the most common complications of diabetes resulting from hyperglycemia leading to ischemic hypoxic tissue and nerve damage. Staphylococcus aureus is the most frequently isolated bacteria from DFUs and causes severe necrotic infections leading to amputations with a poor 5-year survival rate. However, very little is known about the mechanisms by which S. aureus dominantly colonizes and causes severe disease in DFUs. Herein, we utilized a pressure wound model in diabetic TALLYHO/JngJ mice to reproduce ischemic hypoxic tissue damage seen in DFUs and demonstrated that anaerobic fermentative growth of S. aureus significantly increased the virulence and the severity of disease by activating two-component regulatory systems leading to expression of virulence factors. Our in vitro studies showed that supplementation of nitrate as a terminal electron acceptor promotes anaerobic respiration and suppresses the expression of S. aureus virulence factors through inactivation of two-component regulatory systems, suggesting potential therapeutic benefits by promoting anaerobic nitrate respiration. Our in vivo studies revealed that dietary supplementation of L-arginine (L-Arg) significantly attenuated the severity of disease caused by S. aureus in the pressure wound model by providing nitrate. Collectively, these findings highlight the importance of anaerobic fermentative growth in S. aureus pathogenesis and the potential of dietary L-Arg supplementation as a therapeutic to prevent severe S. aureus infection in DFUs.IMPORTANCES. aureus is the most common cause of infection in DFUs, often resulting in lower-extremity amputation with a distressingly poor 5-year survival rate. Treatment for S. aureus infections has largely remained unchanged for decades and involves tissue debridement with antibiotic therapy. With high levels of conservative treatment failure, recurrence of ulcers, and antibiotic resistance, a new approach is necessary to prevent lower-extremity amputations. Nutritional aspects of DFU treatment have largely been overlooked as there has been contradictory clinical trial evidence, but very few in vitro and in vivo modelings of nutritional treatment studies have been performed. Here we demonstrate that dietary supplementation of L-Arg in a diabetic mouse model significantly reduced duration and severity of disease caused by S. aureus. These findings suggest that L-Arg supplementation could be useful as a potential preventive measure against severe S. aureus infections in DFUs.

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.

Ginkgo biloba L. exocarp petroleum ether extract inhibits methicillin-resistant Staphylococcus aureus by modulating ion transport, virulence, and biofilm formation in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: As reported in the Ancient Chinese Medicinal Books, Ginkgo biloba L. fruit has been used as a traditional Chinese medicine for the treatment asthma and cough or as a disinfectant. Our previous study demonstrated that G. biloba exocarp extract (GBEE), an extract of a traditional Chinese herb, inhibits the formation of methicillin-resistant Staphylococcus aureus (MRSA) biofilms. However, GBEE is a crude extract that contains many components, and the underlying mechanisms of purified GBEE fractions extracted with solvents of different polarities are unknown. AIM OF THE STUDY: This study aimed to investigate the different components in GBEE fractions extracted with solvents of different polarities and their antibacterial effects and mechanisms against MRSA and Staphylococcus haemolyticus biofilms both in vitro and in vivo. METHODS: The components in different fractions were detected by high-performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS). Microbroth dilution assays and time growth curves were used to determine the antibacterial effects of the fractions on 15 clinical bacterial isolates. Crystal violet staining, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were utilized to identify the fractions that affected bacterial biofilm formation. The potential MRSA targets of the GBEE fraction obtained with petroleum ether (PE), denoted GBEE-PE, were screened by transcriptome sequencing, and the gene expression profile was verified by quantitative polymerase chain reaction (qPCR). RESULTS: HPLC-HRMS analysis revealed that the four GBEE fractions (extracted with petroleum ether, ethyl acetate, n-butanol, and water) contained different ginkgo components, and the antibacterial effects decreased as the polarity of the extraction solvent increased. The antibacterial activity of GBEE-PE was greater than that of the GBEE fraction extracted with ethyl acetate (EA). GBEE-PE improved H. illucens survival and reduced MRSA colonization in model mouse organs. Crystal violet staining and SEM and TEM analyses revealed that GBEE-PE inhibited MRSA and S. haemolyticus biofilm formation. Transcriptional analysis revealed that GBEE-PE inhibits MRSA biofilms by altering ion transport, cell wall metabolism and virulence-related gene expression. In addition, the LO2 cell viability and H. illucens toxicity assay data showed that GBEE-PE at 20 mg/kg was nontoxic. CONCLUSION: The GBEE fractions contained different components, and their antibacterial effects decreased with increases in the polarity of the extraction solvent. GBEE-PE limited MRSA growth and biofilm formation by affecting ion transport, cell wall synthesis, and virulence-related pathways. This research provides a more detailed overview of the mechanism by which GBEE-PE inhibits MRSA both in vitro and in vivo and suggests that GBEE-PE is a new prospective antimicrobial with the potential to be used in MRSA therapeutics in the future.

Discovery of psoralen as a quorum sensing inhibitor suppresses Pseudomonas aeruginosa virulence.

Pseudomonas aeruginosa is a common opportunistic pathogen with growing resistance and presents heightened treatment challenges. Quorum sensing (QS) is a cell-to-cell communication system that contributes to the production of a variety of virulence factors and is also related to biofilm formation of P. aeruginosa. Compared to traditional antibiotics which kill bacteria directly, the anti-virulence strategy by targeting QS is a promising strategy for combating pseudomonal infections. In this study, the QS inhibition potential of the compounds derived from the Traditional Chinese Medicines was evaluated by using in silico, in vitro, and in vivo analyses. The results showed that psoralen, a natural furocoumarin compound derived from Psoralea corylifolia L., was capable of simultaneously inhibiting the three main QS regulators, LasR, RhlR, and PqsR of P. aeruginosa. Psoralen had no bactericidal activity but could widely inhibit the production of extracellular proteases, pyocyanin, and biofilm, and the cell motilities of the model and clinical P. aeruginosa strains. RNA-sequencing and quantitative PCR analyses further demonstrated that a majority of QS-activated genes in P. aeruginosa were suppressed by psoralen. The supplementation of psoralen could protect Caenorhabditis elegans from P. aeruginosa challenge, especially for the hypervirulent strain PA14. Moreover, psoralen showed synergistic antibacterial effects with polymyxin B, levofloxacin, and kanamycin. In conclusions, this study identifies the anti-QS and antibiofilm effects of psoralen against P. aeruginosa strains and sheds light on the discovery of anti-pseudomonal drugs among Traditional Chinese Medicines. KEY POINTS: • Psoralen derived from Psoralea corylifolia L. inhibits the virulence-related phenotypes of P. aeruginosa. • Psoralen simultaneously targets the three core regulators of P. aeruginosa QS system and inhibits the expression of a large part of downstream genes. • Psoralen protects C. elegans from P. aeruginosa challenge and enhances the susceptibility of P. aeruginosa to antibiotics.

Metabolomics responses and tolerance of Pseudomonas aeruginosa under acoustic vibration stress.

Sound has been shown to impact microbial behaviors. However, our understanding of the chemical and molecular mechanisms underlying these microbial responses to acoustic vibration is limited. In this study, we used untargeted metabolomics analysis to investigate the effects of 100-Hz acoustic vibration on the intra- and extracellular hydrophobic metabolites of P. aeruginosa PAO1. Our findings revealed increased levels of fatty acids and their derivatives, quinolones, and N-acylethanolamines upon sound exposure, while rhamnolipids (RLs) showed decreased levels. Further quantitative real-time polymerase chain reaction experiments showed slight downregulation of the rhlA gene (1.3-fold) and upregulation of fabY (1.5-fold), fadE (1.7-fold), and pqsA (1.4-fold) genes, which are associated with RL, fatty acid, and quinolone biosynthesis. However, no alterations in the genes related to the rpoS regulators or quorum-sensing networks were observed. Supplementing sodium oleate to P. aeruginosa cultures to simulate the effects of sound resulted in increased tolerance of P. aeruginosa in the presence of sound at 48 h, suggesting a potential novel response-tolerance correlation. In contrast, adding RL, which went against the response direction, did not affect its growth. Overall, these findings provide potential implications for the control and manipulation of virulence and bacterial characteristics for medical and industrial applications.

Identification and pathogenicity of Fusarium spp. associated with tea wilt in Zhejiang Province, China.

BACKGROUND: Tea is one of the most widely consumed beverages in the world, with significant economic and cultural value. However, tea production faces many challenges due to various biotic and abiotic stresses, among which fungal diseases are particularly devastating. RESULTS: To understand the identity and pathogenicity of isolates recovered from tea plants with symptoms of wilt, phylogenetic analyses and pathogenicity assays were conducted. Isolates were characterized to the species level by sequencing the ITS, tef-1α, tub2 and rpb2 sequences and morphology. Four Fusarium species were identified: Fusarium fujikuroi, Fusarium solani, Fusarium oxysporum, and Fusarium concentricum. The pathogenicity of the Fusarium isolates was evaluated on 1-year-old tea plants, whereby F. fujikuroi OS3 and OS4 strains were found to be the most virulent on tea. CONCLUSIONS: To the best of our knowledge, this is the first report of tea rot caused by F. fujikuroi in the world. This provides the foundation for the identification and control of wilt disease in tea plants.

Transcriptomic analysis of Vibrio alginolyticus challenged by Rhizoma coptidis reveals mechanisms of virulence genes.

Rhizoma coptidis, a Chinese herbal medicine widely used to treat various bacterial infections, has the potential to develop antibiotic substitutes to overcome the drug resistance of Vibrio alginolyticus. To study the inhibitory effect of R. coptidis on V. alginolyticus, we sequenced the transcriptomes of three groups of samples of wild-type V. alginolyticus (CK) and V. alginolyticus, which were stressed by 5 mg/mL R. coptidis for 2 h (RC_2 h) and 4 h (RC_4 h). CK was compared with RC_2 h and RC_4 h, respectively, and a total of 1565 differentially expressed genes (DEGs) (988 up-regulated and 577 down-regulated) and 1737 DEGs (1152 up-regulated and 585 down-regulated) were identified. Comparing RC_2 h with RC_4 h, 156 DEGs (114 up-regulated and 42 down-regulated) were identified. The ability of biofilm formation and motility of V. alginolyticus altered upon with different concentrations of R. coptidis. Interestingly, relative expression patterns of virulence genes appeared statistically significantly varied, upon different concentrations of R. coptidis extract. DEGs were annotated to the Gene Ontology (GO) database for function enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the results showed that the main enriched pathways, was those related to the virulence of V. alginolyticus. This study provides a new perspective for understanding the complex pathogenic mechanism of V. alginolyticus. R. coptidis could potnetially be used as alternative or complimnetary to antibiotics to treat infections after further research.

The essential role of arginine biosynthetic genes in lunate conidia formation, conidiation, mycelial growth, and virulence of nematophagous fungus, Esteya vermicola CBS115803.

BACKGROUND: The pinewood nematode (Bursaphelenchus xylophilus) causes severe damage to pine trees. The nematophagous fungus, Esteya vermicola, exhibits considerable promise in the biological control of Bursaphelenchus xylophilus due to its infectivity. Notably, the lunate conidia produced by E. vermicola can infect Bursaphelenchus xylophilus. In the study, we aim to investigate the genes involved in the formation of the lunate conidia of E. vermicola CBS115803. RESULTS: Esteya vermicola CBS115803 yielded 95% lunate conidia on the complete medium (CM) and 86% bacilloid conidia on the minimal medium (MM). Transcriptomic analysis of conidia from both media revealed a significant enrichment of differentially expressed genes in the pathway related to 'cellular amino acid biosynthesis and metabolism'. Functional assessment showed that the knockout of two arginine biosynthesis genes (EV232 and EV289) resulted in defects in conidia germination, mycelial growth, lunate conidia formation, and virulence of E. vermicola CBS115803 in Bursaphelenchus xylophilus. Remarkably, the addition of arginine to the MM improved mycelial growth, conidiation and lunate conidia formation in the mutants and notably increased conidia yield and the lunate conidia ratio in the wild-type E. vermicola CBS115803. CONCLUSION: This investigation confirms the essential role of two arginine biosynthesis genes in lunate conidia formation in E. vermicola CBS115803. The findings also suggest that the supplementation of arginine to the culture medium can enhance the lunate conidia yield. These insights contribute significantly to the application of E. vermicola CBS115803 in managing Bursaphelenchus xylophilus infections. © 2023 Society of Chemical Industry.

NIT24 and NIT29-mediated IAA synthesis of Xanthomonas oryzae pv. oryzicola suppresses immunity and boosts growth in rice.

Auxin plays a pivotal role in the co-evolution of plants and microorganisms. Xanthomonas oryzae pv. oryzicola (Xoc) stands as a significant factor that affects rice yield and quality. However, the current understanding of Xoc's capability for indole 3-acetic acid (IAA) synthesis and its mechanistic implications remains elusive. In this study, we performed a comprehensive genomic analysis of Xoc strain RS105, leading to the identification of two nitrilase enzyme family (NIT) genes, designated as AKO15524.1 and AKO15829.1, subsequently named NIT24 and NIT29, respectively. Our investigation unveiled that the deletion of NIT24 and NIT29 resulted in a notable reduction in IAA synthesis capacity within RS105, thereby impacting extracellular polysaccharide production. This deficiency was partially ameliorated through exogenous IAA supplementation. The study further substantiated that NIT24 and NIT29 have nitrilase activity and the ability to catalyse IAA production in vitro. The lesion length and bacterial population statistics experiments confirmed that NIT24 and NIT29 positively regulated the pathogenicity of RS105, suggesting that NIT24 and NIT29 may regulate Xoc invasion by affecting IAA synthesis. Furthermore, our analysis corroborated mutant strains, RS105_ΔNIT24 and RS105_ΔNIT29, which elicited the outbreak of reactive oxygen species, the deposition of callose and the upregulation of defence-related gene expression in rice. IAA exerted a significant dampening effect on the immune responses incited by these mutant strains in rice. In addition, the absence of NIT24 and NIT29 affected the growth-promoting effect of Xoc on rice. This implies that Xoc may promote rice growth by secreting IAA, thus providing a more suitable microenvironment for its own colonization. In summary, our study provides compelling evidence for the existence of a nitrilase-dependent IAA biosynthesis pathway in Xoc. IAA synthesis-related genes promote Xoc colonization by inhibiting rice immune defence response and affecting rice growth by increasing IAA content in Xoc.

Multifunctionality of AsCFEM6 and AsCFEM12 effectors from the potato early blight pathogen Alternaria solani.

Plant pathogens secrete fungal-specific common in several fungal extracellular membrane (CFEM) effectors to manipulate host immunity and contribute to their virulence. Little is known about effectors and their functions in Alternaria solani, the necrotrophic fungal pathogen causing potato early blight. To identify candidate CFEM effector genes, we mined A. solani genome databases. This led to the identification of 12 genes encoding CFEM proteins (termed AsCFEM1-AsCFEM12) and 6 of them were confirmed to be putative secreted effectors. In planta expression revealed that AsCFEM6 and AsCFEM12 have elicitor function that triggers plant defense response including cell death in different botanical families. Targeted gene disruption of AsCFEM6 and AsCFEM12 resulted in a change in spore development, significant reduction of virulence on potato and eggplant susceptible cultivars, increased resistance to fungicide stress, variation in iron acquisition and utilization, and the involvement in 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway. Using maximum likelihood method, we found that positive selection likely caused the polymorphism within AsCFEM6 and AsCFEM12 homologs in different Alternaria spp. Site-directed mutagenesis analysis indicated that positive selection sites within their CFEM domains are required for cell death induction in Nicotiana benthamiana and are critical for response to abiotic stress in yeast. These results demonstrate that AsCFEM effectors possess additional functions beyond their roles in host plant immune response and pathogen virulence.

Roles of crrAB two-component regulatory system in Klebsiella pneumoniae: growth yield, survival in initial colistin treatment stage, and virulence.

OBJECTIVES: Alternation of the colistin resistance-regulating two-component regulatory system (crrAB) is a colistin-resistance mechanism in Klebsiella pneumoniae (K. pneumoniae), but its role in bacteria is not fully understood. METHODS: Twelve colistin-susceptible K. pneumoniae clinical isolates were included in this study: six crrAB-positive and six crrAB-negative. We deleted the crrAB genes from two crrAB-positive isolates and complemented them. We measured the growth yields by determining growth curves in lysogeny broth and minimal media with or without Fe2+. In vitro selection rates for colistin resistance were determined by exposure to colistin, and survival rates against high concentrations of colistin (20 mg/L) at the early stage of growth (20 min) were investigated. Virulence was determined using a serum bactericidal assay and Galleria mellonella larval infection. RESULTS: The presence of crrAB was not associated with colistin resistance and did not increase the in vitro selection rate of colistin resistance after exposure. The growth yield of crrAB-positive isolates was higher in lysogeny broth media and increased when Fe2+ was added to minimal media. The crrAB-positive isolates showed higher survival rates in the early stages of exposure to high colistin concentrations. Decreased serum resistance was identified in the crrAB-deleted mutants. More G. mellonella larvae survived when infected by crrAB-deleted mutants, and higher survival rates of bacteria were identified within the larvae infected with wild-type than crrAB-deletant isolates. CONCLUSION: Through rapid response to external signals, crrAB would provide advantages for K. pneumoniae survival by increasing the final growth yield and initial survival against colistin treatment. This may partly contribute to the bacterial virulence.

Chemical composition, antifungal, and anti-virulence action of the stem bark of Hancornia speciosa Gomes (Apocynaceae) against Candida spp.

ETHNOPHARMACOLOGICAL RELEVANCE: Hancornia speciosa Gomes is a fruit and medicinal species used for treating infectious diseases of the genitourinary system. However, its mechanism of action against microbes is still not fully understood. Infections in the genitourinary system caused by Candida spp. are associated with its fungal resistance and pathogenicity. New plant-derived compounds are an alternative to fight these Candida infections. AIM OF THE STUDY: The objective of this study was to evaluate the anti-Candida effects of extracts of the stem bark of H. speciosa. This research investigated the chemical composition of sulfuric ether (EEHS) and methanolic (MEHS) extracts, their drug-modifying action on fluconazole, and their anti-virulence action on the morphological transition of Candida species. MATERIALS AND METHODS: The extracts (EEHS and MEHS) of the stem bark of H. speciosa were chemically characterized via qualitative phytochemical screening and by liquid chromatography coupled with mass spectrometry (UPLC-MS-ESI-QTOF). The extracts were evaluated regarding their antifungal effects and fluconazole-modifying activity against Candida albicans, Candida krusei, and Candida tropicalis using the broth microdilution method. Additionally, the study evaluated the inhibition of fungal virulence in Candida species through morphological transition assays. RESULTS: The phytochemical screening revealed the presence of anthocyanidins, anthocyanins, aurones, catechins, chalcones, flavones, flavonols, flavanones, leucoanthocyanidins, tannins (condensed and pyrogallic), and xanthones in both extracts of the stem bark of H. speciosa. The UPLC-MS-ESI-QTOF analysis identified the same compounds in both extracts, predominating phenolic compounds. Some compounds were first time recorded in this species: gluconic acid, cinchonain IIb, cinchonain Ib isomer, and lariciresinol hexoside isomers. Most of the intrinsic antifungal activity was observed for the MEHS against C. krusei (IC50: 58.41 µg/mL). At subinhibitory concentrations (MC/8), the EEHS enhanced the action of fluconazole against all Candida strains. The MEHS exhibited greater efficacy than fluconazole inhibiting C. krusei growth. The EEHS completely inhibited hyphae appearance and reduced pseudohyphae formation in C. albicans. CONCLUSION: The stem bark of H. speciosa is a rich source of bioactive compounds, especially phenolic. Phenolic compounds can have important roles in fighting infectious diseases of the genitourinary system, such as candidiasis. The extracts of H. speciosa improved the action of the drug fluconazole against Candida species, inhibited hyphae appearance, and reduced pseudohyphae formation. The results of this study can support the development of new therapeutics against resistant strains of Candida.

Plant-derived virulence arresting drugs as novel antimicrobial agents: Discovery, perspective, and challenges in clinical use.

Antimicrobial resistance (AMR) emerges as a severe crisis to public health and requires global action. The occurrence of bacterial pathogens with multi-drug resistance appeals to exploring alternative therapeutic strategies. Antivirulence treatment has been a positive substitute in seeking to circumvent AMR, which aims to target virulence factors directly to combat bacterial infections. Accumulated evidence suggests that plant-derived natural products, which have been utilized to treat infectious diseases for centuries, can be abundant sources for screening potential virulence-arresting drugs (VADs) to develop advanced therapeutics for infectious diseases. This review sums up some virulence factors and their actions in various species of bacteria, as well as recent advances pertaining to plant-derived natural products as VAD candidates. Furthermore, we also discuss natural VAD-related clinical trials and patents, the perspective of VAD-based advanced therapeutics for infectious diseases and critical challenges hampering clinical use of VADs, and genomics-guided identification for VAD therapeutic. These newly discovered natural VADs will be encouraging and optimistic candidates that may sustainably combat AMR.

Attenuation of quorum sensing regulated virulence functions and biofilm of pathogenic bacteria by medicinal plant Artemisia annua and its phytoconstituent 1, 8-cineole.

The emergence of multidrug resistance (MDR) in bacterial pathogens is a serious public health concern. A significant therapeutic target for MDR infections is the quorum sensing-regulated bacterial pathogenicity. Determining the anti-quorum sensing abilities of certain medicinal plants against bacterial pathogens as well as the in-silico interactions of particular bioactive phytocompounds with QS and biofilm-associated proteins were the objectives of the present study. In this study, 6 medicinal plants were selected based on their ethnopharmacological usage, screened for Anti-QS activity and Artemisia annua leaf extract (AALE) demonstrated pigment inhibitory activity against Chromobacterium violaceum CV12472. Further, the methanol active fraction significantly inhibited the virulence factors (pyocyanin, pyoverdine, rhamnolipid and swarming motility) of Pseudomonas aeruginosa PAO1 and Serratia marcescens MTCC 97 at respective sub-MICs. The inhibition of biofilm was determined using a microtiter plate test and scanning electron microscopy. Biofilm formation was impaired by 70%, 72% and 74% in P. aeruginosa, C. violaceum and S. marcescens, respectively at 0.5xMIC of the extract. The phytochemical content of the extract was studied using GC-MS and 1, 8-cineole was identified as major bioactive compound. Furthermore, 1, 8-cineole was docked with quorum sensing (QS) proteins (LasI, LasR, CviR, and rhlR) and biofilm proteins (PilY1 and PilT). In silico docking and dynamics simulations studies suggested interactions with QS-receptors CviR', LasI, LasR, and biofilm proteins PilY1, PilT for anti-QS activity. Further, 1, 8-cineole demonstrated 66% and 51% reduction in violacein production and biofilm formation, respectively to validate the findings of computational analysis. Findings of the present investigation suggests that 1, 8-cineole plays a crucial role in the QS and biofilm inhibitory activity demonstrated by Artemisia annua extract. RESEARCH HIGHLIGHTS: Artemisia annua leaf extract (AALE) methanol fraction demonstrated broad-spectrum QS and biofilm inhibition Scanning electron microscopy (SEM) confirmed biofilm inhibition Molecular docking and simulation studies suggested positive interactions of 1,8-cineol with QS-receptors and biofilm proteins.

Isolation, identification, and pathogenicity of Pseudomonas glycinae causing ginseng bacterial soft rot in China.

By tissue separation method, tie-back experiment, and hypersensitive response test in potato, strain XJFL-1 was isolated and identified as the pathogen of ginseng bacterial soft rot in Liaoning Provence, China. The morphological characteristics of XJFL-1 were conformed to the Pseudomonads genus. Microbial fatty acid identification showed the principal cellular fatty acid traits of XLFJ-1 corresponded with Pseudomonas spp. API 50CH test results allowed the differentiation of strain XJFL-1 and MS586T from other closely related Pseudomonas species. The molecular identification, including 16S rRNA analysis and multilocus sequence typing (MLST) analysis, showed that XJFL-1 was in the same branch as P. glycinae MS586T. The genome of XJFL-1 was 6,296,473 bp, with an average guanine/cytosine (G + C) content of 60.72 %. Comparative genomics analysis using ANIb and GGDC algorithms indicated that the maximum value was observed between XJFL-1 and P. glycinae MS586T. The above morphological, cell morphology, and molecular biological identification results supported to identification of XJFL-1 as P. glycinae. This is the first report of P. glycinae as the plant pathogen causing ginseng bacterial root rot in China, which complements the biological significance of the species to a certain extent, enriches the pathogens of ginseng bacterial soft rot, and provides a theoretical basis for further investigation.

Immunomodulatory Potential of Fungal Extracellular Vesicles: Insights for Therapeutic Applications.

Extracellular vesicles (EVs) are membranous vesicular organelles that perform a variety of biological functions including cell communication across different biological kingdoms. EVs of mammals and, to a lesser extent, bacteria have been deeply studied over the years, whereas investigations of fungal EVs are still in their infancy. Fungi, encompassing both yeast and filamentous forms, are increasingly recognized for their production of extracellular vesicles (EVs) containing a wealth of proteins, lipids, and nucleic acids. These EVs play pivotal roles in orchestrating fungal communities, bolstering pathogenicity, and mediating interactions with the environment. Fungal EVs have emerged as promising candidates for innovative applications, not only in the management of mycoses but also as carriers for therapeutic molecules. Yet, numerous questions persist regarding fungal EVs, including their mechanisms of generation, release, cargo regulation, and discharge. This comprehensive review delves into the present state of knowledge regarding fungal EVs and provides fresh insights into the most recent hypotheses on the mechanisms driving their immunomodulatory properties. Furthermore, we explore the considerable potential of fungal EVs in the realms of medicine and biotechnology. In the foreseeable future, engineered fungal cells may serve as vehicles for tailoring cargo- and antigen-specific EVs, positioning them as invaluable biotechnological tools for diverse medical applications, such as vaccines and drug delivery.

Molecular Epidemiology and Virulence Gene Analysis of Methicillin-Resistant Staphylococcus aureus Associated with Skin and Soft Tissue Infections in the Shaoxing Region.

Objective: This study aimed to investigate the prevalence, molecular types, and virulence genes of methicillin-resistant Staphylococcus aureus (MRSA) causing skin and soft tissue infections (SSTIs) in the Shaoxing region. Methods: MRSA strains were collected from patients with SSTIs in Shaoxing People's Hospital from January 2019 to December 2019. We conducted SCCmec typing, Staphylococcus protein A (SPA) typing, multilocus sequence typing (MLST), and virulence gene analysis using whole-genome sequencing on all MRSA strains. Results: The detection rate of community-acquired MRSA (CA-MRSA) isolated from SSTI patients in our hospital was 33.3% (6/18). The primary SCCmec types of CA-MRSA strains were IV and V, with IVg(2B) and V(5C2&5) accounting for 16.7% each. Hospital-acquired MRSA (HA-MRSA) strains primarily exhibited SCCmec types IVa(2B) (25.0%), followed by II(2A) (16.7%), V(5C2) (16.7%), and V(5C2&5) (8.3%). SPA typing indicated that CA-MRSA strains causing SSTIs were predominantly t437 (14.3%), t034 (14.3%), t309 (14.3%), t4549 (14.3%), and t7637 (14.3%). The primary SPA type of HA-MRSA strains was t311 (16.7%). MLST typing revealed that the main sequence types (STs) of CA-MRSA strains causing SSTIs were ST22 (33.3%), followed by ST398, ST59, ST88, and ST630, each accounting for 16.7%. The principal STs of HA-MRSA strains were ST398 (16.7%), ST59 (16.7%), ST88 (16.7%), and ST5 (16.7%), followed by ST22, ST630, ST6, and ST188, each at 8.3%. The primary clones of CA-MRSA strains causing SSTIs were ST59-t437-IVg(2B) (16.7%) and ST630-t4549-V(5C2&5) (16.7%), while the primary clones of HA-MRSA strains were ST59-t437-IVa(2B), ST630-t4549-V(5C2&5), ST6-t304-IVa(2B), ST5-t311-II(2A), ST59-t172-IVa(2B), ST398-t571-V(5C2), ST398-t034-V(5C2), and ST5-t311-II(2A), each accounting for 8.3%. The detection rate of the lukSF-PV virulence gene was higher in CA-MRSA strains (50.0%) than in HA-MRSA strains (16.7%). Conclusions: The isolation rate of CA-MRSA strains causing SSTIs was high in Shaoxing People's Hospital, with ST59-t437-IVg(2B) and ST630-t4549-V(5C2&5) being the predominant clones. MRSA strains exhibited multiple virulence genes, with the lukSF-PV gene having a higher detection rate in CA-MRSA strains, signifying its importance as a virulence factor in CA-MRSA.