ABSTRACT Carbapenem-resistant Acinetobacter baumannii (CRAB) is a hospital-acquired pathogen of significant clinical importance. Given the slow progress in antibiotic development, phages have emerged as a novel therapeutic strategy against CRAB infections due to their synergistic effects with antibiotics. This study aims to explore phage-antibiotic combination therapy targeting CRAB, thereby providing a theoretical foundation for its clinical translation. Through virulence assessments in Galleria mellonella larvae and mice, CRAB110 was identified as the most virulent strain among 199 clinical CRAB isolates. Transcription analysis revealed that its enhanced pathogenicity may be associated with dysregulated pro-inflammatory responses. Using CRAB110 as the host bacterium, the newly isolated phage, P2, was selected for further study due to its high lytic efficiency and broad host range. The CRAB110 mutant strain CRAB110-2N, treated with P2, exhibited increased drug susceptibility and attenuated virulence compared to the wild-type strain. Both in vitro and in vivo experiments demonstrated that the combination of P2 with antibiotics achieved superior bacterial clearance compared to phage therapy alone. Furthermore, whole-genome analysis of CRAB110-2N revealed a mutation in its ugd. The constructed CRAB110Δugd showed tolerance to P2; the quorum-sensing gene abaI was associated with phage susceptibility, with the CRAB110ΔabaI strain displaying insensitivity to P2. These findings provide experimental evidence supporting the clinical application of phage-antibiotic combination therapy.IMPORTANCEThis study focused on phage-antibiotic combination therapy targeting carbapenem-resistant Acinetobacter baumannii (CRAB), a priority pathogen designated by the World Health Organization (WHO). A highly virulent strain, CRAB110, was screened from 199 clinical isolates, and a novel, highly lytic phage, P2, was isolated from sewage samples. CRAB110-2N, a mutant strain induced by P2 exposure, displayed increased antibiotic susceptibility and attenuated virulence. Key findings showed that the combination of P2 and meropenem achieved a 99% bacterial clearance rate in vitro and exerted significant synergistic effects in vivo. Genomic analysis identified mutations in the ugd gene, and knockout experiments confirmed its correlation with phage susceptibility. Additionally, the quorum-sensing gene abaI was found to be associated with phage susceptibility in CRAB. Collectively, these results provide critical experimental evidence for the clinical translation of this phage-antibiotic combination therapy.

IntroductionAcinetobacter baumannii (A. baumannii) is a Gram-negative coccobacillus that can cause a variety of infectious diseases. The Quorum Sensing (QS) system is a widespread mechanism regulating group behavior in bacteria, playing a crucial role in the virulence regulation of several Gram-negative pathogenic bacteria. The aim of this study was to investigate the effects of the A. baumannii quorum sensing system on capsule production and pathogenicity.MethodsIn this work, a clinical carbapenem-resistant A. baumannii with mucoid colonies (CRAB110) was used to construct an abaI knockout strain (CRAB110ΔabaI) using CRISPR/Cas9 system. Afterwards, changes in capsule production, bacterial virulence, and host immune responses were evaluated by assessing bacterial phenotypes, physiology, gene expression, and effects on the host.ResultsThe results showed that CRAB110ΔabaI significantly affected capsule production, virulence, and immunity compared with CRAB110. Mice infected with CRAB110ΔabaI exhibited a reduction in inflammation compared to those infected with CRAB110. A comparison of the transcriptomes of CRAB110 and CRAB110ΔabaI identified differentially expressed genes related to polysaccharide synthesis (wza, wzb, wzc), galactose metabolism (GalM), ABC transport, and amino acid metabolism. Mouse lung transcriptomic and cellular Western blot data indicated that quorum sensing (QS) exerts its pro-inflammatory effect through Acinetobacter baumannii by influencing the IL-17 and NF-κB pathways.ConclusionThis study suggests that the abaI gene boosts capsule production and pathogenicity of A. baumannii, providing new insights for clinical management of drug-resistant A. baumannii diseases and a foundation for new antimicrobial drugs.

Aspergillus flavus partitivirus 2 (AfPV2) isolate XC-8 from the fungus Aspergillus flavus strain XC-8 was sequenced and analyzed. AfPV2 contains four segments, dsRNA1 to 4. dsRNA1 is 1907 bp in length with an open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) of 565 amino acids (aa). dsRNA2 is 1936 bp in length with an ORF encoding a putative capsid protein (CP) of 508 aa. dsRNA3 is 1799 bp in length with an ORF encoding a hypothetical protein of 482 aa. dsRNA4 is 1650 bp in length with an ORF encoding a hypothetical protein of 400 aa. Phylogenetic analysis showed that AfPV2 is a member of the genus Alphapartitivirus of the family Partitiviridae. BLASTp analysis showed that AfPV2 isolate XC-8 belongs to the same species as AfPV2 isolate UniPR6, which only has two dsRNA segments (GenBank nos. MZ600060.1 and MZ600061.1). Infection by AfPV2 isolate XC-8 did not cause any obvious significant phenotypic changes in A. flavus.

IntroductionPaenibacillus polymyxa is an essential bio-control bacterium capable of producing numerous antagonistic compounds with potential usefulness. Methicillin-resistant Staphylococcus aureus (MRSA) is a significant bacterial strain that infects hospitals and communities, exhibiting considerable antibiotic resistance and posing a substantial threat to human health, thereby becoming a major bio-safety concern worldwide. The purpose of this study was to investigate the antibacterial properties and mechanisms of the secondary metabolites of P. polymyxa (MEZ6) against MRSA.MethodsThis study used microdilution procedures and growth and bactericidal kinetics studies to investigate the effects of MEZ6 metabolites on MRSA, and reverse-phase high-performance liquid chromatography (HPLC) and mass spectrometry (LC/MC) were used to detect the secondary metabolites of MEZ6.Results and discussionThe results show that MEZ6 secondary metabolites can inhibit MRSA growth, prevent biofilm formation, reduce the expression of virulence genes (agrA, spa, and clf-1), disrupt cell structure, increase membrane permeability, and lead to the accumulation of ROS. Through systematic characterization, MEZ6 metabolites maybe tryptophan-associated fraction (TAF). This study establishes a systematic theoretical framework for the development and application of bacterial metabolites.

The urgent development of novel antibacterial agents to combat antibiotic-resistant Staphylococcus aureus is imperative. Antimicrobial peptides are considered promising alternatives to traditional antibiotics for addressing this pressing issue. This study investigates the antibacterial activities and potential mechanisms of the peptide HfAMP against methicillin-resistant S. aureus (MRSA). The antibacterial activity of HfAMP against MRSA was evaluated using the broth microdilution method, growth curve, and time-kill assay. Its potential mechanism of action was investigated through propidium iodide, laurdan, DiSC3(5), 2’,7’-dichlorodihydrofluorescein diacetate, and ATP quantification assays. To assess the safety and stability of HfAMP, cytotoxicity, hemolysis, and antibacterial activity were examined under different conditions (i.e., temperatures, ions, serum, pH, and enzymes). Furthermore, a mouse skin infection model was employed to evaluate the in vivo efficacy of HfAMP. The results demonstrate that HfAMP exhibits bacteriostatic and bactericidal activity against MRSA, with a minimum inhibitory concentration of 4 µg/mL. It remains stable under a range of pH, temperatures, and serum, although it is susceptible to degradation by pepsin. Mechanistic investigations show that HfAMP compromises bacterial membranes by interacting with membrane components and disrupting the proton motive force, leading to metabolic disturbances. In a mouse model of MRSA-induced skin infection, HfAMP significantly reduced bacterial burden and inflammation in the affected skin tissues. Collectively, these findings indicate that HfAMP possesses potent antibacterial activity against MRSA by compromising bacterial viability and inhibiting virulence, highlighting its potential as a promising candidate for the treatment of MRSA skin-infection.

The rapid increase in carbapenem-resistant Klebsiella pneumoniae (CRKP) infections, along with the cross-resistance of CRKP to other antibiotics, has created an urgent need for novel therapeutic agents. Among the potential options for next-generation antibiotics, antimicrobial peptides (AMPs) show great promise. In this study, we aimed to elucidate the mechanisms underlying the antibacterial activity against CRKP of an antibacterial peptide named Cecropin-4 (Cec4), which we successfully identified previously. Our results demonstrate that Cec4 not only exhibits rapid antibacterial activity but also effectively inhibits and eradicates bacterial biofilm at a low concentration of 8 µg/mL. Additionally, when used in combination with traditional antibiotics, Cec4 enhances their antibacterial effect. Microscopy techniques, including transmission electron microscopy (TEM), confocal laser scanning microscopy, and scanning electron microscopy (SEM), found that Cec4 destroyed bacteria's cell membrane integrity and increased the membrane permeability (flow cytometry instrument technology further characterization of Cec4 against K. pneumoniae bacteria antibacterial effect). Furthermore, in vitro experiments demonstrated that Cec4 binds to bacterial DNA and RNA of CRKP. Moreover, in vivo studies using a mouse skin wound model confirmed the efficacy of Cec4, and transcriptomic analysis shed light on the molecular mechanisms underlying its antibacterial activity. Based on our findings, Cec4 appears to be a promising candidate for combating CRKP infections.IMPORTANCEThe rapid increase in carbapenem-resistant Klebsiella pneumoniae (CRKP) infections and the serious cross-resistance to multiple antibiotics make the development of new therapeutic drugs urgent. Antimicrobial peptides (AMPs) have attracted much attention as a potential option for the next generation of antibiotics. Previous studies have identified the antimicrobial peptide Cecropin-4 (Cec4), and this study further explored its antimicrobial mechanism against CRKP. Studies have found that Cec4 shows high antibacterial activity at low concentrations, can inhibit and eradicate bacterial biofilms, and can also enhance the efficacy of traditional antibiotics. Its mechanism of action, such as destroying cell membranes and binding nucleic acid, has been revealed by various techniques, and its effectiveness has been confirmed in vivo, providing a promising candidate drug for combating CRKP infection.

There is a pressing need to create innovative alternative treatment approaches considering the overuse of antifungal drugs causes the number of clinically isolated fluconazole-resistant Candida species to increase. Glycine max antimicrobial peptide (GmAMP) is a novel peptide screened by us using artificial intelligence modeling techniques, and pre-tests showed its strong antimicrobial activity against clinically fluconazole-resistant Candida tropicalis. The study aimed to comprehensively investigate the antimicrobial activity and mechanisms of GmAMP against fluconazole-resistant C. tropicalis. The antifungal activity of GmAMP against fluconazole-resistant C. tropicalis was assessed by using broth microdilution method, growth and fungicidal kinetics, hypha transformation, and antibiofilm assay. To further uncover the potential mechanisms of action of GmAMP, we performed scanning electron microscopy, flow cytometry, cell membrane potential probe 3, 3'-Dipropylthiadicarbocyanine Iodide (DiSC3(5)), and reactive oxygen species (ROS) probe 2', 7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) detection to assess the cellular morphology and structure, membrane permeability, membrane depolarization, and ROS accumulation, respectively. At the same time, we used cytotoxicity and degree of erythrocyte hemolysis assays to assess GmAMP's toxicity in vitro. Cytotoxicity and treatment efficacy were evaluated in vivo by utilizing the Galleria mellonella larvae infection model. GmAMP exhibited significant antifungal activity against fluconazole-resistant C. tropicalis with a minimum inhibitory concentration (MIC) of 25 µM and demonstrated fungicidal effects at 100 µM within 2 h. GmAMP prevented the transition from yeast to hypha morphology, inhibited the biofilm formation rate of 88.32%, and eradicated the mature biofilm rate of 58.28%. Additionally, GmAMP treatment at 100 µM caused cell structure damage in fluconazole-resistant C. tropicalis, whereas GmAMP treatment at concentrations ranging from 25 to 100 µM caused membrane permeability, depolarization of cell membrane potential, and intracellular ROS accumulation. Moreover, GmAMP enhanced the survival rate of 75% for G. mellonella with fluconazole-resistant C. tropicalis infection as well as reduced fungal burden in vivo by approximately 1.0 × 102 colony forming units per larva (CFU per larva). GmAMP can disrupt the cell membrane of fluconazole-resistant C. tropicalis and also shows favorable safety and therapeutic efficacy in vivo. Accordingly, GmAMP has the potential to be an agent against drug-resistant fungi.

IntroductionAcinetobacter baumannii (A. baumannii) is an important opportunistic pathogen causing nosocomial infection in the clinic. The occurrence rate of antibiotic resistance is increasing year by year, resulting in a highly serious situation of bacterial resistance.MethodsTo better understand the local epidemiology of multidrug-resistant A. baumannii, an investigation was conducted on the antibiotic resistance of different types of A. baumannii and its relationship with the genes of A. baumannii. Furthermore, the molecular mechanism underlying antibiotic resistance in A. baumannii was investigated through transcriptome analysis.ResultsThese results showed that a total of 9 STs were detected. It was found that 99% of the strains isolated in the hospital belonged to the same STs, and the clone complex CC208 was widely distributed in various departments and all kinds of samples. Furthermore, these A. baumannii strains showed high resistance to ertapenem, biapenem, meropenem, and imipenem, among which the resistance to ertapenem was the strongest. The detection rate of blaOXA–51 gene in these carbapenem resistance A. baumannii (CRAB) reached 100%; Additionally, the transcriptome results showed that the resistance genes were up-regulated in resistance strains, and these genes involved in biofilm formation, efflux pumps, peptidoglycan biosynthesis, and chaperonin synthesis.DiscussionThese results suggest that the CC208 STs were the main clonal complex, and showed high carbapenem antibiotic resistance. All these resistant strains were distributed in various departments, but most of them were distributed in intensive care units (ICU). The blaOXA–23 was the main antibiotic resistance genotype; In summary, the epidemic trend of clinical A. baumannii in Guiyang, China was analyzed from the molecular level, and the resistance mechanism of A. baumannii to carbapenem antibiotics was analyzed with transcriptome, which provided a theoretical basis for better control of A. baumannii.

Multidrug-resistant (MDR) bacteria pose serious threats to public health due to the lack of effective and biocompatible drugs to kill MDR bacteria. Photodynamic antibacterial therapy has been widely studied due to its low induction of resistance. However, photosensitizers that can efficiently generate reactive oxygen species (ROS) through both type I and type II mechanisms and that have the capability of multiple modes of action are rarely reported. Addressing this issue, we developed a near-infrared-emitting triphenylamine indole iodoethane (TTII) and its silver(I) self-assembled (TTIIS) aggregation-induced emission (AIE) photosensitizer for multimode bacterial infection therapy. TTII can efficiently produce both Type I ROS •OH and Type II ROS 1O2. Interestingly, the Ag(I)-π interaction contributed in TTIIS efficiency promotion of the generation of 1O2. Moreover, by releasing Ag+, TTIIS enabled photodynamic-Ag(I) dual-mode sterilization. As a result, TTIIS achieved an effective enhancement of antibacterial activity, with a 1-2-fold boost against multidrug-resistant Escherichia coli (MDR E. coli). Both TTII and TTIIS at a concentration as low as 0.55 μg mL-1 can kill more than 98% of methicillin resistant Staphylococcus aureus (MRSA) on MRSA-infected full-thickness defect wounds of a mouse, and both TTII and TTIIS were effective in eliminating the bacteria and promoting wound healing.

IntroductionBacterial infections have become serious threats to human health, and the excessive use of antibiotics has led to the emergence of multidrug-resistant (MDR) bacteria. E. coli is a human bacterial pathogen, which can cause severe infectious. Antimicrobial peptides are considered the most promising alternative to traditional antibiotics.Materials and methodsThe minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and hemolytic activity were determined by the microdilution method. The antimicrobial kinetics of MR-22 against E. coli were studied by growth curves and time-killing curves. The cytotoxicity of MR-22 was detected by the CCK-8 assay. The antimicrobial activity of MR-22 in salt, serum, heat and trypsin was determined by the microdilution method. The antimicrobial mechanism of MR-22 against drug-resistant E. coli was studied by Scanning Electron Microscope, laser confocal microscopy, and Flow Cytometry. The in vivo antibacterial activity of MR-22 was evaluated by the mice model of peritonitis.Results and discussionIn this study, MR-22 is a new antimicrobial peptide with good activity that has demonstrated against MDR E. coli. The antimicrobial activity of MR-22 exhibited stability under conditions of high temperature, 10% FBS, and Ca2+. However, a decline of the activity was observed in the presence of Na+, serum, and trypsin. MR-22 had no significant cytotoxicity or hemolysis in vitro. SEM and fluorescent images revealed that MR-22 could disrupt the integrity of cell membrane. DCFH-DA indicated that MR-22 increased the content of reactive oxygen species, while it decreased the content of intracellular ATP. In mice model of peritonitis, MR-22 exhibited potent antibacterial activity in vivo. These results indicated that MR-22 is a potential drug candidate against drug-resistant E. coli.

Candida albicans, a common commensal and opportunistic fungal pathogen in humans, can occasionally progress to disseminated candidiasis which is a serious condition with a high morbidity and fatality rate. The emergence of drug-resistant fungal strains compels us to look for an efficient treatment solution. Our earlier studies have demonstrated that the unique antimicrobial peptide AMP-17 from Musca domestica has a strong antifungal impact on C. albicans in vitro. Here, we verified the therapeutic effects of AMP-17 on systemic candidiasis in vivo and the peptide interacts with fluconazole, a common antifungal medication, to treat systemic candidiasis. In the disseminated candidiasis model of Galleria mellonella and mice challenged with C. albicans, AMP-17 increased the survival rates of infected larvae and mice to 66.7 and 75%, respectively. Furthermore, the peptide lowered the load of C. albicans in the infected larvae and the kidneys of the mice by nearly 90%. Additional histological examination and measurements of plasma cytokines showed that the injection of AMP-17 markedly reduced the inflammatory response and balanced cytokine expression. Furthermore, checkerboard micro dilution experiments demonstrated that AMP-17 and fluconazole worked in synergy to inhibit C. albicans in the biofilm mode. According to morphological studies, AMP-17 and fluconazole together decreased the production of hyphae throughout the C. albicans biofilm formation process, loosening the mature biofilms’ structure and lowering the amount of carbohydrates in the extracellular matrix (ECM) of the biofilms. Taken together, these results showed that AMP-17 would be a viable treatment for systemic candidiasis and might be a different approach to combating Candida biofilm, either by itself or in conjunction with fluconazole.

Purpose:The emergence of carbapenem-resistant Acinetobacter baumannii (CRAB) poses great difficulties in clinical treatment, and has been listed by the World Health Organization as a class of pathogens in urgent need of new antibiotic development. In our previous report, the novel antimicrobial peptide Cec4 showed great potential in decreasing the clinical CRAB biofilm, but its mechanism of action is still illusive. Therefore, in order to evaluate the clinical therapeutic potential of Cec4, it is necessary to explore the mechanism of how Cec4 decreases mature biofilms. Methods:Key genes involved in the removal of CRAB biofilms by Cec4 were analyzed using transcriptomics. Based on the results of the bioinformatics analysis, the CRISPR-Cas9 method was used to construct the deletion strain of the key gene. The pYMAb2 plasmid was used for the complementation strain construction. Finally, the roles of key genes in biofilm removal by Cec4 were determined by crystal violet staining, podocyte staining, laser confocal imaging, and MBC and MBEC50. Results:Combined with transcriptome analysis, we hypothesized that OmpH is a key gene involved in the removal of CRAB biofilms by Cec4. Deletion of the OmpH gene did not affect A. baumannii growth, but decreased A. baumannii capsule thickness, increasing biofilm production, and made biofilm-state A. baumannii more sensitive to Cec4. Conclusion:Cec4 decreases biofilms formed by CRAB targeting OmpH. Deletion of the OmpH gene results in an increase in biofilms and greater sensitivity to Cec4, which enhances the removal of A. baumannii biofilms by Cec4.

Carbapenem-resistant Acinetobacter baumannii (CRAB), an important opportunistic pathogen, is a major cause of healthcare-associated infections. The polymyxins (colistin and polymyxin B) are the last line of defense in the treatment of CRAB infections, and there is an urgent need to develop novel alternative therapeutic strategies. In this study, we found that the antimicrobial peptide DvAMP exhibited satisfactory antibacterial and antibiofilm activity against CRAB. In addition, DvAMP showed tolerable stability in salt ions and serum and exhibited low toxicity in vivo. Investigation of the underlying mechanism demonstrated that DvAMP disrupts cell membrane structural integrity and specifically binds to exogenous lipopolysaccharides (LPS) and phospholipids (PG/CL), resulting in increased membrane permeability and dissipating proton motive force (PMF), further reducing intracellular ATP levels and inducing ROS accumulation, leading to bacterial death. Furthermore, DvAMP therapy efficiently improved survival rates and decreased the bacterial load in the lungs of mice in a mouse pneumonia model, showing that DvAMP administration reduced CRAB susceptibility to lung infection. These results indicate that the peptide DvAMP is a promising alternative therapeutic agent to combat CRAB infection.

The comprehensive antitumor mechanisms of elemene have been discussed in the previous chapter. The current findings on liver cancer, lung cancer, gastric cancer, glioma, prostate cancer, pancreatic cancer, and colorectal cancer are presented in this chapter in detail. The studies reveal the function and mechanism of elemene in inhibiting cancer cell proliferation, inducing apoptosis, arresting cell cycle, inhibiting migration and invasion, inhibiting angiogenesis, promoting senescence, inducing differentiation, and inhibiting epithelial-mesenchymal transition based on the experiments in vivo and in vitro. Moreover, they illustrate that elemene can be combined and is compatible with chemotherapy drugs, targeted drugs, and vaccines to enhance the antitumor efficacy and reverse drug resistance.

Cryptococcus neoformans is an important opportunistic human fungal pathogen that causes cryptococcosis in immunocompromised patients. However, the number of drugs for the treatment of cryptococcosis is restricted, and the development of novel antifungal drugs and innovative strategies for the treatment of cryptococcosis is urgently needed. In this study, we validated that DvAMP is a novel antimicrobial peptide with antimicrobial activity and that it was obtained by pre-screening from the UniProt database of more than three million unknown functional sequences based on the quantitative structure-activity relationships (QSARs) protocol (http://www.chemoinfolab.com/antifungal). The peptide exhibited satisfactory biosafety and physicochemical properties, and relatively rapid fungicidal activity against C. neoformans. Meanwhile, DvAMP was able to inhibit the static biofilm of C. neoformans and cause a reduction in the thickness of the capsule. In addition, DvAMP exerts antifungal effects through membrane-mediated mechanisms (membrane permeability and depolarization) and mitochondrial dysfunction, involving a hybrid multi-hit mechanism. Furthermore, by using the C. neoformans-Galleria mellonella infection model, we demonstrated that DvAMP has significant therapeutic effects in vivo and that it significantly reduces the mortality and fungal burden of infected larvae. These results suggest that DvAMP may be a potential antifungal drug candidate for the treatment of cryptococcosis.

Bladder cancer (BLCA) typically has a poor prognosis due to high rates of relapse and metastasis. Although the emergence of immunotherapy brings hope for patients with BLCA, not all patients will benefit from it. Identifying some markers to predict treatment response is particularly important. Here, we aimed to determine the clinical value of the ribonuclease/angiogenin inhibitor 1 (RNH1) in BLCA therapy based on functional status analysis. First, we found that RNH1 is aberrantly expressed in multiple cancers but is associated with prognosis in only a few types of cancer. Next, we determined that low RNH1 expression was significantly associated with enhanced invasion and metastasis of BLCA by assessing the relationship between RNH1 and 17 functional states. Moreover, we identified 95 hub genes associated with invasion and metastasis among RNH1-related genes. Enrichment analysis revealed that these hub genes were also significantly linked with immune activation. Consistently, BLCA can be divided into two molecular subtypes based on these hub genes, and the differentially expressed genes between the two subtypes are also significantly enriched in immune-related pathways. This indicates that the expression of RNH1 is also related to the tumour immune response. Subsequently, we confirmed that RNH1 shapes an inflammatory tumour microenvironment (TME), promotes activation of the immune response cycle steps, and has the potential to predict the immune checkpoint blockade (ICB) treatment response. Finally, we demonstrated that high RNH1 expression was significantly associated with multiple therapeutic signalling pathways and drug targets in BLCA. In conclusion, our study revealed that RNH1 could provide new insights into the invasion of BLCA and predict the immunotherapy response in patients with BLCA.

Antimicrobial peptides are potential alternatives to traditional antibiotics in the face of increasing bacterial resistance. Insects possess many antimicrobial peptides and have become a valuable source of novel and highly effective antimicrobial peptides. Hermetia illucens as a resource insect, for example, has the highest number of antimicrobial peptides of any dipteran. However, most antimicrobial peptides, especially cecropin, have not been comprehensively identified and have not been evaluated for their antimicrobial ability. In this study, we analyzed the localization and gene structure of 33 cecropin molecules in the H. illucens genome and evaluated their activity against common human pathogens. The results showed that 32 cecropin molecules were concentrated on 1 chromosome, most with 2 exons. More importantly, most of the cecropins had a good antibacterial effect against Gram-negative bacteria, and were not hemolytic. The minimum inhibitory concentration (MIC) of the cecropin designated H3 against E. coli was 4 μg/mL. The toxicity, killing time kinetics, and anti-biofilm activity of H3 were further investigated and confirmed its antimicrobial ability. Overall, H3 is a potential candidate for the development of new antimicrobials to treat severe infections caused by Gram-negative pathogens such as E. coli.

Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti-Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall (XOG1) and oxidative stress (SRR1) genes upon the action of AMP-17 on C. albicans, suggesting that AMP-17 may exert anti-C. albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C. albicansxog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C. albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C. albicans. Drug sensitivity assay showed that the MIC80 values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 μg/mL (for the wild type C. albicans SC5314) to 16 μg/mL, while the MIC80 values against srr1Δ/Δ: : srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti-C. albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.

A double-stranded RNA (dsRNA) mycovirus was obtained from Aspergillus terreus strain HJ3-26 and designated "Aspergillus terreus chrysovirus 1" (AtCV1). It consists of four dsRNA segments (dsRNA1-4) with lengths of 3612 bp, 3132 bp, 3153 bp, and 3144 bp, respectively. Sequence analysis showed that dsRNA1 encodes an RNA-dependent RNA polymerase (RdRp), dsRNA2 encodes a capsid protein, and both dsRNA3 and dsRNA4 encode hypothetical proteins. Phylogenetic analysis of the RdRp suggested that AtCV1 is a member of a new species of the genus Alphachrysovirus in the family Chrysoviridae. This is the first chrysovirus obtained from A. terreus.

There is a need for new anti-Candida albicans (C. albicans) drugs owing to the emergence of drug resistance in recent years. AMP-17, an antimicrobial peptide from Musca domestica (M. domestica), is known to be an effective inhibitor of many fungal pathogens, including C. albicans. In this study, we investigated the potential mechanism underlying the anti-C. albicans effects of AMP-17 using flow cytometry, transmission electron microscopy, fluorescent probes, fluorescence microplate reader, and confocal laser microscopy. Transmission electron microscopy showed that, following AMP-17 treatment, the shape of C. albicans cells became irregular, and vacuoles could be seen in the cytoplasm. Furthermore, AMP-17 treatment resulted in an increase in reactive oxygen species (ROS) levels, depolarization of the mitochondrial membrane potential (MMP), and changes in the cell cycle, leading to the apoptosis and necrosis, which ultimately contributed to the death of C. albicans cells.