Antifungal and Antivirulence Activity of Vanillin and Tannic Acid Against Aspergillus fumigatus and Fusarium solani.

Aspergillus fumigatus and Fusarium solani infections have become severe health threat; both pathogens are considered a priority due to the increasing emergence of antifungal-resistant strains and high mortality rates. Therefore, the discovery of new therapeutic strategies has become crucial. In this study, we evaluated the antifungal and antivirulence effects of vanillin and tannic acid against Aspergillus fumigatus and Fusarium solani. The minimum inhibitory concentrations of the compounds were determined by the microdilution method in RPMI broth in 96-well microplates according to CLSI. Conidial germination, protease production, biofilm formation, and in vivo therapeutic efficacy assays were performed. The results demonstrated that vanillin and tannic acid had antifungal activity against Aspergillus fumigatus, while tannic acid only exhibited antifungal activity against Fusarium solani. We found that vanillin and tannic acid inhibited conidial germination and secreted protease production and biofilm formation of the fungal pathogens using sub-inhibitory concentrations. Besides, vanillin and tannic acid altered the fungal membrane permeability, and both compounds showed therapeutic effect against aspergillosis and fusariosis in an infection model in Galleria mellonella larvae. Our results highlight the antivirulence effect of vanillin and tannic acid against priority pathogenic fungi as a possible therapeutic alternative for human fungal infections.

Naphthylisoquinoline alkaloids: novel agents against the causative pathogens of eumycetoma and actinomycetoma-en route to broad-spectrum antimycetomal drugs.

Mycetoma is a devastating neglected tropical infection of the subcutaneous tissues. It is caused by fungal and bacterial pathogens recognized as eumycetoma and actinomycetoma, respectively. Mycetoma treatment involves diagnosing the causative microorganism as a prerequisite to prescribing a proper medication. Current therapy of fungal eumycetoma causative agents, such as Madurella mycetomatis, consists of long-term antifungal medication with itraconazole followed by surgery, yet with usually unsatisfactory clinical outcomes. Actinomycetoma, on the contrary, usually responds to treatment with co-trimoxazole and amikacin. Therefore, there is a pressing need to discover novel broad-spectrum antimicrobial agents to circumvent the time-consuming and costly diagnosis. Using the resazurin assay, a series of 23 naphthylisoquinoline (NIQ) alkaloids and related naphthoquinones were subjected to in vitro screening against two fungal strains of M. mycetomatis and three bacterial strains of Actinomadura madurae and A. syzygii. Seven NIQs, mostly dimers, showed promising in vitro activities against at least one strain of the mycetoma-causative pathogens, while the naphthoquinones did not show any activity. A synthetic NIQ dimer, 8,8'''-O,O-dimethylmichellamine A (18), inhibited all tested fungal and bacterial strains (IC50 = 2.81-12.07 µg/mL). One of the dimeric NIQs, michellamine B (14), inhibited a strain of M. mycetomatis and significantly enhanced the survival rate of Galleria mellonella larvae infected with M. mycetomatis at concentrations of 1 and 4 µg/mL, without being toxic to the uninfected larvae. As a result, broad-spectrum dimeric NIQs like 14 and 18 with antimicrobial activity are considered hit compounds that could be worth further optimization to develop novel lead antimycetomal agents.

Pathogenicity of Metarhizium rileyi (Hypocreales: Clavicipitaceae) against Tenebrio molitor (Coleoptera: Tenebrionidae).

Tenebrio molitor L., also known as the mealworm, is a polyphagous insect pest that infests various stored grains worldwide. Both the adult and larval stages can cause significant damage to stored grains. The present study focused on isolating entomopathogenic fungi from an infected larval cadaver under environmental conditions. Fungal pathogenicity was tested on T. molitor larvae and pupae for 12 days. Entomopathogenic fungi were identified using biotechnological methods based on their morphology and the sequence of their nuclear ribosomal internal transcribed spacer (ITS). The results of the insecticidal activity indicate that the virulence of fungi varies between the larval and pupal stages. In comparison to the larval stage, the pupal stage is highly susceptible to Metarhizium rileyi, exhibiting 100% mortality rates after 12 days (lethal concentration 50 [LC50] = 7.8 × 106 and lethal concentration 90 (LC90) = 2.1 × 1013 conidia/mL), whereas larvae showed 92% mortality rates at 12 days posttreatment (LC50 = 1.0 × 106 and LC90 = 3.0 × 109 conidia/mL). The enzymatic analyses revealed a significant increase in the levels of the insect enzymes superoxide dismutase (4.76-10.5 mg-1) and glutathione S-transferase (0.46-6.53 mg-1) 3 days after exposure to M. rileyi conidia (1.5 × 105 conidia/mL) compared to the control group. The findings clearly show that M. rileyi is an environmentally friendly and effective microbial agent for controlling the larvae and pupae of T. molitor.

Differential induction of NF-κB pathways by non-pathogenic and pathogenic bacteria in Helicoverpa armigera is critical for an efficient immune response and survival.

Following pathogen infection in a host, extensive changes occur in the host's gene expression pattern to suppress infection and increase the chance of host survival. Likewise, many pathogens have evolved to evade/suppress host immunity and increase their survival within the host. In this study, we assessed the NF-κB (Imd and Toll) essential gene expression response of Helicoverpa armigera to an entomopathogenic Serratia marcescens and non-pathogenic Escherichia coli. Bacterial cells of S. marcescens or E. coli were injected into the haemocoel of fifth-instar larvae of H. armigera, whereas distilled water was injected into control insects. Our results showed that the expression levels of the Imd and Toll pathway genes (i.e., Relish, imd, spätzle and dif) and the antimicrobial peptides (i.e., gloverin, transferin, gallerimycin, and galiomicin) were differentially expressed following the bacterial injections while control larvae showed no differences. The E. coli injection induced higher and longer-lasted gene expression than the S. marcescens injected larvae, in which the gene expressions were diminished from 24 h post injection. Transcript Knockdown of Relish increased the replication rates of S. marcescens and E. coli, and lowered the infected larvae survival rates. These results showed that H. armigera NF-κB immunity pathways (particularly Imd pathway) play a vital role in immunity against bacterial infections, and S. marcescens might modulate these pathways to survive and replicate in the host.

Infection-experienced HSPCs protect against infections by generating neutrophils with enhanced mitochondrial bactericidal activity.

Hematopoietic stem and progenitor cells (HSPCs) respond to infection by proliferating and generating in-demand neutrophils through a process called emergency granulopoiesis (EG). Recently, infection-induced changes in HSPCs have also been shown to underpin the longevity of trained immunity, where they generate innate immune cells with enhanced responses to subsequent microbial threats. Using larval zebrafish to live image neutrophils and HSPCs, we show that infection-experienced HSPCs generate neutrophils with enhanced bactericidal functions. Transcriptomic analysis of EG neutrophils uncovered a previously unknown function for mitochondrial reactive oxygen species in elevating neutrophil bactericidal activity. We also reveal that driving expression of zebrafish C/EBPß within infection-naïve HSPCs is sufficient to generate neutrophils with similarly enhanced bactericidal capacity. Our work suggests that this demand-adapted source of neutrophils contributes to trained immunity by providing enhanced protection toward subsequent infections. Manipulating demand-driven granulopoiesis may provide a therapeutic strategy to boost neutrophil function and treat infectious disease.

Impact of Beauveria bassiana on antioxidant enzyme activities and metabolomic profiles of Spodoptera frugiperda.

Spodoptera frugiperda is a pest that poses a serious threat to the production of food and crops. Entomopathogenic fungi, such as Beauveria bassiana, have shown potential for S. frugiperda control. However, the mechanism of this biological control of pathogens is not fully understood, such as how antioxidant enzyme activities and metabolic profiles in S. frugiperda larvae are affected when infected by entomopathogenic fungi. This study assessed the antioxidant enzyme activities and shift in metabolomic profile in the S. frugiperda larvae infected with B.bassiana. The results indicate a pattern of initial increase and subsequent decrease in the activities of superoxide dismutase, catalase, and peroxidase in the B.bassiana-infected larvae. And the enzyme activities at 60 h of infection ended significantly lower than those of the uninfected larvae. A total of 93 differential metabolites were identified in the B.bassiana-infected larvae, of which 41 metabolites were up-regulated and 52 were down-regulated. These metabolites mainly included amino acids, nucleotides, lipids, carbohydrates, and their derivatives. Among the changed metabolites, cystathionine, l-tyrosine, l-dopa, arginine, alpha-ketoglutaric acid, d-sedoheptulose-7-phosphate and citric acid were significantly decreased in B. bassiana-infected larvae. This indicated that the fungal infection might impair the ability of S. frugiperda larvae to cope with oxidative stress, leading to a negative impact of organism fitness. Further analyses of key metabolic pathways reveal that B. bassiana infection might affect purine metabolism, arginine biosynthesis, butanoate metabolism, and phenylalanine metabolism of S. frugiperda larvae. The findings from this study will contribute to our understanding of oxidative stress on immune defense in insects, and offer fundamental support for the biological control of S. frugiperda.

Helicoverpa zea-Associated Gut Bacteria as Drivers in Shaping Plant Anti-herbivore Defense in Tomato.

Insect-associated bacteria can mediate the intersection of insect and plant immunity. In this study, we aimed to evaluate the effects of single isolates or communities of gut-associated bacteria of Helicoverpa zea larvae on herbivore-induced defenses in tomato. We first identified bacterial isolates from the regurgitant of field-collected H. zea larvae by using a culture-dependent method and 16S rRNA gene sequencing. We identified 11 isolates belonging to the families Enterobacteriaceae, Streptococcaceae, Yersiniaceae, Erwiniaceae, and unclassified Enterobacterales. Seven different bacterial isolates, namely Enterobacteriaceae-1, Lactococcus sp., Klebsiella sp. 1, Klebsiella sp. 3, Enterobacterales, Enterobacteriaceae-2, and Pantoea sp., were selected based on their phylogenetic relationships to test their impacts on insect-induced plant defenses. We found that the laboratory population of H. zea larvae inoculated with individual isolates did not induce plant anti-herbivore defenses, whereas larvae inoculated with a bacterial community (combination of the 7 bacterial isolates) triggered increased polyphenol oxidase (PPO) activity in tomato, leading to retarded larval development. Additionally, field-collected H. zea larvae with an unaltered bacterial community in their gut stimulated higher plant defenses than the larvae with a reduced gut microbial community. In summary, our findings highlight the importance of the gut microbial community in mediating interactions between herbivores and their host plants.

Microbacterium tenebrionis sp. nov. and Microbacterium allomyrinae sp. nov., isolated from larvae of Tenebrio molitor L. and Allomyrina dichotoma, respectively.

Two Gram-positive bacterial strains, designated as YMB-B2T and BWT-G7T, were isolated from larvae of Tenebrio molitor L. and Allomyrina dichotoma, respectively, and their taxonomic positions examined by a polyphasic approach. Both of the isolates contained ornithine as the cell-wall diamino acid. The acyl type of murein was N-glycolyl. The predominant menaquinones were MK-11 and MK-12. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. Both of the isolates contained C15 : 0 anteiso and C17 : 0 anteiso as the major fatty acids. Strain YMB-B2T also had C16 : 0 iso as an additional major fatty acid. The 16S rRNA gene phylogeny showed that the novel strains formed two distinct sublines within the genus Microbacterium. Strain YMB-B2T was most closely related to the type strains of Microbacterium aerolatum (99.1 % sequence similarity) and Microbacterium ginsengiterrae (99.0 %), whereas strain BWT-G7T formed a tight cluster with the type strain of Microbacterium thalassium (98.9 %). The phylogenomic analysis based on 92 core genes supported their relationships in 16S rRNA gene phylogeny. Overall genomic relatedness indices warranted that the isolates represent two new species of the genus Microbacterium. Based on the results obtained here, Microbacterium tenebrionis sp. nov. (type strain YMB-B2T=KCTC 49593T=CCM 9151T) and Microbacterium allomyrinae sp. nov. (type strain BWT-G7T=KACC 22262T=NBRC 115127T) are proposed.

Tomato Chemical Defenses Intensify Corn Earworm (Helicoverpa zea) Mortality from Opportunistic Bacterial Pathogens.

Insect herbivores face multiple challenges to their ability to grow and reproduce. Plants can produce a series of defenses that disrupt and damage the herbivore digestive system, which are heightened upon injury by insect feeding. Additionally, insects face threats from virulent microorganisms that can incur their own set of potential costs to hosts. Microorganisms that invade through the digestive system may function in concert with defenses generated by plants, creating combined assailments on host insects. In our study, we evaluated how tomato defenses interact with an enteric bacterial isolate, Serratia marcescens, in the corn earworm (Helicoverpa zea). We performed bioassays using different tomato cultivars that were induced by methyl jasmonate and larvae orally inoculated with a S. marcescens isolate. Untreated corn earworm larval mortality was low on constitutive tomato, while larvae inoculated with S. marcescens exhibited > 50% mortality within 5 days. Induction treatments elevated both control mortality (~ 45%) and in combination with S. marcescens (> 95%). Larvae also died faster when encountering induced defenses and Serratia. Using a tomato mutant, foliar polyphenol oxidase activity likely had stronger impacts on S. marcescens-mediated larval mortality. Induction treatments also elevated the number of bacterial colony-forming units in the hemolymph of larvae inoculated with Serratia. Larval mortality by S. marcescens was low (< 10%) on artificial diets. Our results demonstrate that plant chemical defenses enhance larval mortality from an opportunistic gut microbe. We propose that the combined damage from both the plant and microbial agent overwhelm the herbivore to increase mortality rates and expedite host death.

Effect of acute ultraviolet radiation on Galleria mellonella health and immunity.

For humans, acute and chronic overexposure to ultraviolet (UV) radiation can cause tissue damage in the form of sunburn and promote cancer(s). The immune-modulating properties of UV radiation and health-related consequences are not well known. Herein, we used the larvae of the wax moth Galleria mellonella, to determine UV-driven changes in cellular components of innate immunity. From immune cell (haemocyte) reactivity and the production of antimicrobial factors, these insects share many functional similarities with mammalian cellular innate immunity. After exposing insects to UVA or UVB for up to two hours, we monitored larval viability, susceptibility to infection, haemolymph (blood) physiology and faecal discharge. Prolonged exposure of larvae to UVB coincided with decreased survival, enhanced susceptibility to bacterial challenge, melanin synthesis in the haemolymph, compromised haemocyte functionality and changes in faecal (bacterial) content. We contend G. mellonella is a reliable in vivo model for assessing the impact of UV exposure at the whole organism and cellular levels.

Growth of Acinetobacter baumannii impacted by iron chelation.

Acinetobacter baumannii (AB) has become multidrug-resistant (MDR) in recent years, and, currently, there are limited effective treatment options. Nutrient metals (e.g. iron) are essential to the metabolic functions of AB. This study examined the impact of iron chelation on the growth of AB in vitro and in vivo. Susceptible and MDR-AB bloodstream isolates (n = 9) were recovered from different patients between 2011 and 2018. Clonal diversity was ascertained by Fourier-transform infrared spectroscopy. In vitro bacterial densities were measured over 20 h to determine growth profiles. Variable amounts of a chelating agent [deferiprone (DFP)] were added to create a concentration gradient. Galleria mellonella larvae were inoculated with an isolate, with and without DFP. Quantitative culture was used to ascertain the bacterial burden of aggregate larvae immediately and 4 h post-infection. Increasing concentrations of DFP caused a transient and concentration-dependent hindrance to in vitro growth, compared to the no-treatment group. In vivo bacterial burden immediately post-infection in both groups was comparable. After 4 h, the burden was much higher in the control group comparatively (8.7 and 6.7 log CFU g-1). These results support that micro-nutrient limitation has the potential of being a novel approach for treating high-risk infections due to MDR-AB.

Physiological and Molecular Response Modifications by Ultraviolet-C Radiation in Plutella xylostella and Its Compatibility with Cordyceps fumosorosea.

Ultraviolet-C (UV-C) radiation significantly impacts living organisms. UV-C radiation can also be used as a pest management tool. Therefore, this study was designed to investigate the effect of UV-C radiation on the physiology and gene expression level of Plutella xylostella, a destructive vegetable pest. Results showed that, after exposure to UV-C radiation for 3, 6, 12, and 24 h, the activity of SOD (superoxide dismutase) and CAT (catalase) of P. xylostella increased, while the activity of PPO (polyphenol oxidase), POD (peroxidase), AChE (acetylcholinesterase), CarE (carboxylesterase), and ACP (acid phosphatase) decreased with increased exposure time. Correlation coefficient analyses indicated that the activity of CAT correlated positively, while PPO and CarE correlated negatively, with exposure time. Gene regulation analysis via qRT-PCR confirmed a significant increase in regulation in CAT, CarE, and PPO-related genes. We also investigated the effect of UV-C exposure on the virulence of Cordyceps fumosorosea against P. xylostella. Here, results indicated that when the fungal treatment was applied to larvae before UV-C radiation, the virulence of C. fumosorosea was significantly reduced. However, this decline in virulence of C. fumosorosea due to UV-C exposure remained only for one generation, and no effect was observed on secondary infection. On the other hand, when larvae were exposed to UV-C radiation before fungal application, the mortality rate significantly increased as the exposure time to UV-C radiation increased. From the current study, it could be concluded that UV-C exposure suppressed the immunity to P. xylostella, which later enhanced the virulence of entomopathogenic fungi. Moreover, the study also suggested that UV irradiation is an effective pest management tool that could be incorporated into pest management strategies, which could help reduce pesticide application, be economically beneficial for the farmer, and be environmentally safe.

Entomopathogenic fungi and Schinus molle essential oil: The combination of two eco-friendly agents against Aedes aegypti larvae.

Aedes aegypti transmits arbovirus, which is a public health concern. Certain filamentous fungi have the potential to control the disease. Here, the effects of Metarhizium anisopliae s.l. CG 153, Beauveria bassiana s.l. CG 206 and Schinus molle L. were investigated against Aedes aegypti larvae. In addition, the effect of essential oil on fungal development was analyzed. Fungal germination was assessed after combination with essential oil at 0.0025 %, 0.0075 %, 0.005 %, or 0.01 %; all of the oil concentrations affected germination except 0.0025 % (v/v). Larvae were exposed to 0.0025 %, 0.0075 %, 0.005 %, or 0.01 % of the essential oil or Tween 80 at 0.01 %; however, only the essential oil at 0.0025 % achieved similar results as the control. Larvae were exposed to fungi at 107 conidia mL-1 alone or in combination with the essential oil at 0.0025 %. Regardless of the combination, M. anisopliae reduced the median survival time of mosquitoes more than B. bassiana. The cumulative survival of mosquitoes exposed to M. anisopliae alone or in combination with essential oil was 7.5 % and 2 %, respectively, and for B. bassiana, it was 75 % and 71 %, respectively. M. anisopliae + essential oil had a synergistic effect against larvae, whereas B. bassiana + essential oil was antagonistic. Scanning and transmission electron microscopy, and histopathology confirmed that the interaction of M. anisopliae was through the gut and hemocoel. In contrast, the mosquito's gut was the main route for invasion by B. bassiana. Results from gas chromatography studies demonstrated sabinene and bicyclogermacrene as the main compounds of S. molle, and the in-silico investigation found evidence that both compounds affect a wide range of biological activity. For the first time, we demonstrated the potential of S. molle and its interaction with both fungal strains against A. aegypti larvae. Moreover, for the first time, we reported that S. molle might be responsible for significant changes in larval physiology. This study provides new insights into host-pathogen interplay and contributes to a better understanding of pathogenesis in mosquitoes, which have significant consequences for biological control strategies.

Insecticidal activities of the local entomopathogenic nematodes and cell-free supernatants from their symbiotic bacteria against the larvae of fall webworm, Hyphantriacunea.

The fall webworm (FWW), Hyphantria cunea Drury (Lepidoptera: Erebidae), is an invasive and polyphagous insect pest of many economically important crops such as hazelnuts, apple, and mulberry. Recently, there have been an increasing number of reports about the damaging activities of FWW from hazelnut growing areas of Turkey indicating that currently existing control methods fail to satisfy the expectations of growers. Entomopathogenic nematodes (EPNs) in the Steinernematidae and Heterorhabditidae (Nematoda: Rhabditida) families and the symbiotic bacteria they carry in their intestine have a great potential for the management of many agriculturally important pests. In this study, the symbiotic bacteria of local EPN species (Heterorhabditis bacteriophora AVB-15, Steinernema feltiae KCS-4S, and Steinernema bicornotum MGZ-4S) recovered from the central Anatolia region was characterized using recA gene region as Photorhabdus luminescens, Xenorhabdus bovienii and Xenorhabdus budapestensis. The contact (25, 50, 100, 200 IJs/Petri) and oral efficacies of the infective juveniles (IJs) (25, 50, 100, 200 IJs/leaf) of these EPN isolates determined on 3rd/4th instar larvae, and cell-free supernatants from the identified symbiotic bacteria were evaluated separately on the 3rd and 4th larval instars of FWW in Petri dish environment under laboratory conditions (25 ± 1 °C, 60% of RH). In the Petri dish bioassays of EPN species, the most pathogenic isolate at the 1st DAT and 4th DAT was S. feltiae which caused 50% mortality at the highest concentration (200 IJs/Petri) and the highest mortality rate (97.5%) were achieved at 4th DAT by H. bacteriophora AVB-15 isolate. Surprisingly, the mortality rates were generally higher at the lowest concentrations and 82.5% mortality were reached 4th DAT by S. bicornotum at the lowest concentration (25 IJs/leaf) in the leaf bioassays. Mortality rates were higher in both Petri dish and filter paper efficacies of cell-free supernatants at the 2nd DAT and the highest mortality (87.5%) was reached in the contact efficacy studies when applied X. bovienii KCS-4S strain. The results suggest that the tested EPN species and CFSs have good potential for biological control of the larvae of FWW and can contribute to the IPM programs of FWW. However, the efficacy of both IJs of EPNs and CFSs of their symbiotic bacteria on larvae of FWW requires further studies to verify their efficiency in the field.

Biocontrol potential of cell suspensions and cell-free superntants of different Xenorhabdus and Photorhabdus bacteria against the different larval instars of Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae).

The black cutworm (BCW), Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), is one of the destructive cutworm species. Black cutworm is a highly polyphagous pest that feeds on more than 30 plants, many of which are of economic importance such as maize, sugar beet, and potato. The control of BCW larvae relies heavily on the application of synthetic insecticides which have a detrimental impact on human health and the natural environment. In addition, increasing insecticide resistance in many insect species requires a novel and sustainable approach to controlling insect pests. The endosymbionts of entomopathogenic nematodes (EPNs) (Xenorhabdus and Phorohabdus spp.) represent a newly emerging green approach to controlling a wide range of insect pests. In the current study, the oral and contact efficacy of cell suspension (4 × 107 cells ml-1) and cell-free supernatants of different symbiotic bacteria (X. nematophilai, X. bovienii, X. budapestensis, and P. luminescent subsp. kayaii) were evaluated against the mixed groups of 1st-2nd and 3rd-4th instars larvae of BCW under controlled conditions. The oral treatment of the cell suspension and cell-free supernatants resulted in higher mortality rates than contact treatments. In general, larval mortality was higher in the 1st-2nd instar larvae than in the 3rd-4th instar larvae. The highest (75%) mortality was obtained from the cell suspension of X. budapestensis. The results indicated that the oral formulations of the cell suspension and cell-free supernatants of bacterial strains may have a good control potential against the 1st-2nd larvae BCW. However, the efficacy of the cell suspension and cell-free supernatants of tested bacterial strains should be further evaluated under greenhouse and field conditions.

Nitric Oxide-Induced Calcineurin A Mediates Antimicrobial Peptide Production Through the IMD Pathway.

Nitric oxide (NO) at a high concentration is an effector to kill pathogens during insect immune responses, it also functions as a second messenger at a low concentration to regulate antimicrobial peptide (AMP) production in insects. Drosophila calcineurin subunit CanA1 is a ubiquitous serine/threonine protein phosphatase involved in NO-induced AMP production. However, it is unclear how NO regulates AMP expression. In this study, we used a lepidopteran pest Ostrinia furnacalis and Drosophila S2 cells to investigate how NO signaling affects the AMP production. Bacterial infections upregulated the transcription of nitric oxide synthase 1/2 (NOS1/2), CanA and AMP genes and increased NO concentration in larval hemolymph. Inhibition of NOS or CanA activity reduced the survival of bacteria-infected O. furnacalis. NO donor increased NO level in plasma and upregulated the production of CanA and certain AMPs. In S2 cells, killed Escherichia coli induced NOS transcription and boosted NO production, whereas knockdown of NOS blocked the NO level increase caused by E. coli. As in O. furnacalis larvae, supplementation of the NO donor increased NO level in the culture medium and AMP expression in S2 cells. Suppression of the key pathway genes showed that the IMD (but not Toll) pathway was involved in the upregulation of CecropinA1, Defensin, Diptericin, and Drosomycin by killed E. coli. Knockdown of NOS also reduced the expression of CanA1 and AMPs induced by E. coli, indicative of a role of NO in the AMP expression. Furthermore, CanA1 RNA interference and inhibition of its phosphatase activity significantly reduced NO-induced AMP expression, and knockdown of IMD suppressed NO-induced AMP expression. Together, these results suggest that NO-induced AMP production is mediated by CanA1 via the IMD pathway.

Biological control potential of entomopathogenic fungal strains against peach Fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae).

The peachfruit fly, Bactrocera zonata (Saunders) is a polyphagous pest in nature, belonging to order, Diptera and their respective family is Tephritidae. It mostly feeds on different crops, vegetables and fruits. Different traditional chemical insecticides have been used to control this notorious pest. Excessive consumption of pesticides has become a major threat to the fresh fruits trade since many importing countries refused to accept the shipments due to public health and environmental concerns. There is a growing trend to control these pests using the most effective biological control methods and other preventive measures have been adopted for reducing their attacks. Fungal agents have been used as biological agents to manage the attack of different insects pest through biological means. The present study was conducted to assess the virulence of three entomopathogenic fungi, Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii, against Bactrocera zonata stages under different laboratory conditions. The results showed that B. bassiana and M. anisopliae were more effective in pathogenicity and potentially kill at all stages of B. zonata as compared to V. lecanii. The highest mortality rate for the third larval instar and the pupal stage were recorded after exposure to the 1 × 1010 conidia/ml concentrations, B. bassiana, with 68.67% and 89.67%, respectively. Adult B. zonata flies were the most susceptible to all entomopathogenic fungi. However, M. anisopliae was more virulent against B. zonata adult flies than B. bassiana and V. lecanii at 1 × 1010 conidial concentration. Therefore, the entomopathogenic fungi B. bassiana and M. anisopliae can be used as an cost effective bio-insecticide in the integrated pest management programs to control B. zonata. This study will be helpful to overcome this pest through biological control means.

Emergence of polymyxin B-heteroresistant hypervirulent Klebsiella pneumoniae from an individual in the community with asymptomatic bacteriuria.

BACKGROUND: The heteroresistance of polymyxin B, a last-resort antibiotic used to treat many serious bacterial infections, may lead to antibiotic treatment failure. However, polymyxin B-heteroresistant isolates are rare in individuals living in the community. We report a polymyxin B-heteroresistant hypervirulent Klebsiella pneumoniae (hvKP) isolate from an individual in the community with asymptomatic bacteriuria. RESULTS: The NYTJ35 isolate had multiple virulence genes that encoded a mucoid phenotype regulator (rmpA), aerobactin (iucABCD-iutA), salmochelin (iroBCDN), yersiniabactin (irp1-2 and ybtAEPQSTUX), and a truncated rmpA2. Infection of galleria mellonella larvae indicated the isolate was hypervirulent. Antimicrobial susceptibility testing showed it was susceptible to all tested antibiotics except polymyxin B. The proportion of surviving bacteria was 1.2 × 10- 7 based on the population analysis profile (PAP) method, suggesting the presence of polymyxin B heteroresistance. The isolate was not hypermucoviscous, but it was a strong biofilm producer. It had capsular serotype K1 and belonged to sequence type 23 (ST23). The isolate also had the D150G substitution in phoQ, which is known to confer polymyxin B resistance. CONCLUSIONS: We identified the co-occurrence of hypervirulence and polymyxin B heteroresistance in a K. pneumoniae isolate from an individual with asymptomatic bacteriuria. We suggest the use of increased screening for hvKP in individuals living in the community.

WMR Peptide as Antifungal and Antibiofilm against Albicans and Non-Albicans Candida Species: Shreds of Evidence on the Mechanism of Action.

Candida species are the most common fungal pathogens infecting humans and can cause severe illnesses in immunocompromised individuals. The increased resistance of Candida to traditional antifungal drugs represents a great challenge in clinical settings. Therefore, novel approaches to overcome antifungal resistance are desired. Here, we investigated the use of an antimicrobial peptide WMR against Candida albicans and non-albicans Candida species in vitro and in vivo. Results showed a WMR antifungal activity on all Candida planktonic cells at concentrations between 25 µM to >50 µM and exhibited activity at sub-MIC concentrations to inhibit biofilm formation and eradicate mature biofilm. Furthermore, in vitro antifungal effects of WMR were confirmed in vivo as demonstrated by a prolonged survival rate of larvae infected by Candida species when the peptide was administered before or after infection. Additional experiments to unravel the antifungal mechanism were performed on C. albicans and C. parapsilosis. The time-killing curves showed their antifungal activity, which was further confirmed by the induced intracellular and mitochondrial reactive oxygen species accumulation; WMR significantly suppressed drug efflux, down-regulating the drug transporter encoding genes CDR1. Moreover, the ability of WMR to penetrate within the cells was demonstrated by confocal laser scanning microscopy. These findings provide novel insights for the antifungal mechanism of WMR against Candida albicans and non-albicans, providing fascinating scenarios for the identification of new potential antifungal targets.

In vitro and in vivo antifungal activity of two peptides with the same composition and different distribution.

Candida albicans can cause local or systemic diseases when host immune status is disrupted. Drug resistance to C. albicans highlights the necessity of novel antifungal drugs. Antimicrobial peptides exhibit potential as antifungal drugs. PAF26 was found to exhibit favorable activity against plant pathogenic fungi. However, it showed low antifungal activity against C. albicans. Here, P255 and P256 with the same composition and different distribution were derived from PAF26. P256 exhibited higher antifungal activity against C. albicans than did P255 and PAF26. P256 and P255 exhibited synergism when combined with amphotericin B (AMB). Both peptides reduced cell wall integrity, rapidly increased membrane permeability, disrupted cell morphology and intracellular alterations. The peptides affected the expression of fungal DNA replication and repair, cell wall synthesis and ergosterol synthesis genes. They increased cellular reactive oxygen species production and bound with fungal genomic DNA. Antibiofilm activities were observed when peptide alone or combined with AMB. Finally, these peptides protected 70% of Galleria mellonella from infection-caused death. Insects treated with peptides exhibited fewer infection foci compared with the untreatment. These results demonstrate the therapeutic potential of the peptides, particularly P256 with clear amphipathicity, in the development of therapies for C. albicans infections.