Interactions between Bacillus thuringiensis and selected plant extracts for sustainable management of Phthorimaea absoluta.

Phthorimaea absoluta is a global constraint to tomato production and can cause up to 100% yield loss. Farmers heavily rely on synthetic pesticides to manage this pest. However, these pesticides are detrimental to human, animal, and environmental health. Therefore, exploring eco-friendly, sustainable Integrated Pest Management approaches, including biopesticides as potential alternatives, is of paramount importance. In this context, the present study (i) evaluated the efficacy of 10 Bacillus thuringiensis isolates, neem, garlic, and fenugreek; (ii) assessed the interactions between the most potent plant extracts and B. thuringiensis isolates, and (iii) evaluated the gut microbial diversity due to the treatments for the development of novel formulations against P. absoluta. Neem recorded the highest mortality of 93.79 ± 3.12% with an LT50 value of 1.21 ± 0.24 days, Bt HD263 induced 91.3 ± 3.68% mortality with LT50 of 2.63 ± 0.11 days, compared to both Bt 43 and fenugreek that caused < 50% mortality. Larval mortality was further enhanced to 99 ± 1.04% when Bt HD263 and neem were combined. Furthermore, the microbiome analyses showed that Klebsiella, Escherichia and Enterobacter had the highest abundance in all treatments with Klebsiella being the most abundant. In addition, a shift in the abundance of the bacterial genera due to the treatments was observed. Our findings showed that neem, garlic, and Bt HD263 could effectively control P. absoluta and be integrated into IPM programs after validation by field efficacy trials.

Biofilm Inhibition of Inula viscosa (L.) Aiton and Globularia alypum L. Extracts Against Candida Infectious Pathogens and In Vivo Action on Galleria mellonella Model.

The increasing importance of fungal infections has fueled the search for new beneficial alternatives substance from plant extracts. The current study investigates the antifungal and antibiofilm activity of Inula viscosa (L.) Aiton and Globularia alypum (L.) leaves extracts against Candida both in vitro and in vivo. The inhibition of planktonic and sessile Candida albicans and Candida glabrata growth using both leaf extracts are evaluated. Moreover; an in vivo infection model using Galleria mellonella larvae; infected and treated with the extracts are performed. All extracts show fungicidal activity; with a minimum fungicidal concentration (MFC) ranging from 128 to 512 µg mL-1 against the two selected strains of Candida. In particular, the best results are obtained with methanolic extract of I. viscosa and G. alypum with an MFC value of 128 µg mL-1 . The extracts are capable to prevent 90% of biofilm development at minor concentrations ranging from 100.71 ± 2.49 µg mL-1 to 380.4 ± 0.92 µg mL-1 . In vivo, tests on Galleria mellonella larvae show that the extracts increase the survival of the larvae infected with Candida. The attained results reveal that I. viscosa and G. alypum extracts may be considered as new antifungal agents and biofilm inhibiting agents for the pharmaceutical and agro-food field.

Co-diet supplementation of low density polyethylene and honeybee wax did not influence the core gut bacteria and associated enzymes of Galleria mellonella larvae (Lepidoptera: Pyralidae).

The current plastic pollution throughout the world is a rising concern that demands the optimization of biodegradation processes. One avenue for this is to identify plastic-degrading bacteria and associated enzymes from the gut bacteria of insect models such as Tenebrio molitor, Plodia interpunctella or Galleria mellonella that have the ability to ingest and rapidly degrade polyethylene. Therefore, this study takes part in understanding the role of the gut bacteria by investigating G. mellonella as a biological model feeding with a diet based on honeybee wax mixed or not with low-density polyethylene. Gut microbiome was analyzed by high throughput 16S rRNA sequencing, and Enterococcaceae and Oxalobacteraceae were found to be the major bacterial families. Compared to the control, the supplementation of low-density polyethylene did not cause significant modification of the bacterial microbiota at community and taxa levels, suggesting bacterial microbiome resilience. The bacterial proteome analysis of gut contents was encouraging for the identification of plastic degrading enzymes such as the phenylacetaldehyde dehydrogenase which participate in styrene degradation. This study allowed a better characterization of the gut bacteria of G. mellonella and provided a basis for the further study of biodegradation of polyethylene based on the bacterial microbiota from insect guts.

Screening the pandemic response box identified benzimidazole carbamates, Olorofim and ravuconazole as promising drug candidates for the treatment of eumycetoma.

Eumycetoma is a chronic subcutaneous neglected tropical disease that can be caused by more than 40 different fungal causative agents. The most common causative agents produce black grains and belong to the fungal orders Sordariales and Pleosporales. The current antifungal agents used to treat eumycetoma are itraconazole or terbinafine, however, their cure rates are low. To find novel drugs for eumycetoma, we screened 400 diverse drug-like molecules from the Pandemic Response Box against common eumycetoma causative agents as part of the Open Source Mycetoma initiative (MycetOS). 26 compounds were able to inhibit the growth of Madurella mycetomatis, Madurella pseudomycetomatis and Madurella tropicana, 26 compounds inhibited Falciformispora senegalensis and seven inhibited growth of Medicopsis romeroi in vitro. Four compounds were able to inhibit the growth of all five species of fungi tested. They are the benzimidazole carbamates fenbendazole and carbendazim, the 8-aminoquinolone derivative tafenoquine and MMV1578570. Minimal inhibitory concentrations were then determined for the compounds active against M. mycetomatis. Compounds showing potent activity in vitro were further tested in vivo. Fenbendazole, MMV1782387, ravuconazole and olorofim were able to significantly prolong Galleria mellonella larvae survival and are promising candidates to explore in mycetoma treatment and to also serve as scaffolds for medicinal chemistry optimisation in the search for novel antifungals to treat eumycetoma.

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding.

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here, we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse ß-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

Antibiotics, like penicillin, are the foundation of modern medicine, but bacteria are evolving to resist their effects. Some of the most harmful pathogens belong to a group called the 'Gram-negative bacteria', which have an outer layer ­ called the cell envelope ­ that acts as a drug barrier. This envelope contains antibiotic resistance proteins that can deactivate or repel antibiotics or even pump them out of the cell once they get in. One way to tackle antibiotic resistance could be to stop these proteins from working. Proteins are long chains of building blocks called amino acids that fold into specific shapes. In order for a protein to perform its role correctly, it must fold in the right way. In bacteria, a protein called DsbA helps other proteins fold correctly by holding them in place and inserting links called disulfide bonds. It was unclear whether DsbA plays a role in the folding of antibiotic resistance proteins, but if it did, it might open up new ways to treat antibiotic resistant infections. To find out more, Furniss, Kaderabkova et al. collected the genes that code for several antibiotic resistance proteins and put them into Escherichia coli bacteria, which made the bacteria resistant to antibiotics. Furniss, Kaderabkova et al. then stopped the modified E. coli from making DsbA, which led to the antibiotic resistance proteins becoming unstable and breaking down because they could not fold correctly. Further experiments showed that blocking DsbA with a chemical inhibitor in other pathogenic species of Gram-negative bacteria made these bacteria more sensitive to antibiotics that they would normally resist. To demonstrate that using this approach could work to stop infections by these bacteria, Furniss, Kaderabkova et al. used Gram-negative bacteria that produced antibiotic resistance proteins but could not make DsbA to infect insect larvae. The larvae were then treated with antibiotics, which increased their survival rate, indicating that blocking DsbA may be a good approach to tackling antibiotic resistant bacteria. According to the World Health Organization, developing new treatments against Gram-negative bacteria is of critical importance, but the discovery of new drugs has ground to a halt. One way around this is to develop ways to make existing drugs work better. Making drugs that block DsbA could offer a way to treat resistant infections using existing antibiotics in the future.

Extracts of Sida cordifolia contain polysaccharides possessing immunomodulatory activity and rosmarinic acid compounds with antibacterial activity.

BACKGROUND: The overuse of antibiotics has led to increased antimicrobial resistance, but plant-derived biological response modifiers represent a potential alternative to these drugs. This investigation examined the immunomodulatory and antibacterial activities of Sida cordifolia (used in ethnomedicinal systems to treat infectious disease). METHODS: Successive extractions were performed from the roots of these plants in hexane, chloroform, methanol and water. Immunomodulatory activity was determined in a series of experiments measuring the responses of splenocytes, macrophages and an in vivo model of innate immunity (Galleria mellonella). Antibacterial activity was assessed by determining minimum inhibitory/bactericidal concentrations (MIC/MBCs) for various Gram-positive and Gram-negative bacterial strains. RESULTS: Immunomodulatory activity was confined to the aqueous extract, and further fractionation and biochemical analysis yielded a highly potent polysaccharide-enriched fraction (SCAF5). SCAF5 is a complex mixture of different polysaccharides with multiple immunomodulatory effects including immune cell proliferation, antibody secretion, phagocytosis, nitric oxide production, and increased expression of pro-inflammatory cytokines. Furthermore, Galleria mellonella pre-treated with SCAF5 produced more haemocytes and were more resistant (P < 0.001) to infection with methicillin-resistant Staphylococcus aureus (MRSA) with a 98% reduction in bacterial load in pre-treated larvae compared to the negative control. The antibacterial activity of Sida cordifolia was confined to the methanolic fraction. Extensive fractionation identified two compounds, rosmarinic acid and its 4-O-ß-d-glucoside derivative, which had potent activity against Gram-positive antibiotic-resistant bacteria, including MRSA. CONCLUSIONS: Sida cordifolia counters bacterial infections through a dual mechanism, and immunomodulatory polysaccharides from this plant should be isolated and characterised to realise their potential as anti-infective agents. Such properties could be developed as an antibiotic alternative (1) in the clinic and (2) alternative growth promoter for the agri-food industry.

Deep Sequencing, Nested PCR, and Denaturing Gradient Gel Electrophoresis Reveal a Wider Distribution of Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes), in Native Soil Types.

Ophiocordyceps sinensis appears as stroma emerging from underground sclerotium enclosed by the skeleton of Thitarodes moth larvae. However, the actual distribution of the fungus in soil still remains unclarified. In this study, 40 soil samples were used for detection of O. sinensis to confirm its distribution in native habitats using denaturing gradient gel electrophoresis, nested internal transcribed spacer (ITS) PCR, and 454 pyrosequencing methods. The soil samples included six types: Os, where both stromata and host moth larvae were found; NL, representing no signs of stromata, but where moth larvae were found; NOs, where neither stroma nor moth larvae were found; BS, with bare soil without the presence of stroma of O. sinensis or moth larvae; AF, from soil surrounding the stroma; and MP, soil particles firmly wrapping the sclerotium of O. sinensis. Of 40 samples tested, 36 showed positive detection of O. sinensis by at least one of the three detection methods, with positive detection in all six sample types at all five sites. The results showed that traces of O. sinensis can be detected in locations with no macroscopically visible evidence of the fungus or its host and at least 100 m away from such locations.

In Vitro and In Vivo Biological Activity of Berberine Chloride against Uropathogenic E. coli Strains Using Galleria mellonella as a Host Model.

Berberine is an alkaloid of the protoberberine type used in traditional oriental medicine. Its biological activities include documented antibacterial properties against a wide variety of microorganisms; nonetheless, its use against Escherichia coli strains isolated from urinary infections has not yet been widely investigated in vivo. The emergence of antimicrobial resistance requires new therapeutic approaches to ensure the continued effectiveness of antibiotics for the treatment and prevention of urinary infections. Moreover, uropathogenic Escherichia coli (UPEC) has developed several virulence factors and resistance to routine antibiotic therapy. To this end, several in vitro and in vivo tests were conducted to assess the activity of berberine on uropathogenic E. coli strains. Galleria mellonella as an infection model was employed to confirm the in vivo translatability of in vitro data on berberine activity and its influence on adhesion and invasion proprieties of E. coli on human bladder cells. In vitro pre-treatment with berberine was able to decrease the adhesive and invasive UPEC ability. In vivo treatment increased the larvae survival infected with UPEC strains and reduced the number of circulating pathogens in larvae hemolymph. These preliminary findings demonstrated the efficacy and reliability of G. mellonella as in vivo model for pre-clinical studies of natural substances.

Bacteriophage-antibiotic combinations against ciprofloxacin/ceftriaxone-resistant Escherichia coli in vitro and in an experimental Galleria mellonella model.

Escherichia coli is the most common cause of Gram-negative prosthetic joint infections (PJIs) and ciprofloxacin is the first-line antibiofilm antibiotic. Due to the emergence of fluoroquinolone resistance, management of E. coli PJIs has become challenging and is associated with high treatment failure rates. We evaluated the efficacy of a newly isolated bacteriophage ɸWL-3 as a therapeutic agent in combination with ciprofloxacin, fosfomycin, gentamicin, meropenem or ceftriaxone against biofilm of a ciprofloxacin/ceftriaxone-resistant E. coli strain and the ATCC 25922 reference strain. ɸWL-3 was first characterised in terms of virion morphology, absorption rate, burst size and killing kinetics against both E. coli strains. The tested antibiotics presented high inhibitory concentrations (ranging from 16 to >1024 µg/mL) when tested alone against biofilms. Co-administration of ɸWL-3 with antibiotics improved the antibiotic efficacy against biofilm, especially after staggered exposure, reducing the minimum biofilm bactericidal concentration (MBBC) up to 512 times. The in vivo antimicrobial activity of ɸWL-3/fosfomycin combination against both E. coli strains was assessed in a Galleria mellonella invertebrate infection model. Treatment of infected larvae after lethal doses of E. coli resulted in enhanced survival rates when combinatorial therapy with ɸWL-3/fosfomycin was applied on E. coli ATCC 25922-infected larvae compared with monotherapy, but not for EC1-infected larvae, which we speculated could be due to higher release of endotoxins in a shorter period in EC1-infected larvae exposed to ɸWL-3. Our study provides new insights into the use of bacteriophages and antibiotics in the treatment of biofilm-associated infections caused by antibiotic-resistant bacteria.

Immunomodulatory and Antioxidant Effects of Polysaccharides from the Parasitic Fungus Cordyceps kyushuensis.

The ascomycete Cordyceps genus has been used as valued traditional Chinese medicine. Cordyceps kyushuensis is a unique species of Cordyceps, which parasitizes on the larvae of Clanis bilineata Walker, and its major component cordycepin and aqueous extract are known to have many pharmacological effects. However, the physiological function of water-soluble polysaccharides has not been explored in detail. In this study, to resolve these doubts, we extracted and separated Cordyceps-derived polysaccharides and then evaluated the immunomodulatory and antioxidant activities. Four polysaccharide fractions were purified from Cordyceps-cultured stroma by DEAE-cellulose 23 and Sephadex G-150 column chromatography. Basic structural information was elucidated on the basis of physicochemical property and spectroscopic evidences. The antioxidant activities were evaluated by a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method and protective effect of DNA damage. The qualified immunologic activities were also determined in vivo and in vitro. The polysaccharides could stimulate the proliferation of mouse splenocytes whether concanavalin A (ConA) and lipopolysaccharide (LPS) existed or not, strengthen peritoneal macrophages to devour neutral red, and increase the content of interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) in serum. The research provides the corresponding evidence for Cordyceps polysaccharides as a potential candidate for functional foods and therapeutic agents.

The combined utilization of Chlorhexidine and Voriconazole or Natamycin to combat Fusarium infections.

BACKGROUND: Fusarium species are the fungal pathogens most commonly responsible for the mycotic keratitis, which are resistant to the majority of currently available antifungal agents. The present study was designed to assess the efficacy of a combination of low doses chlorhexidine with two other commonly used drugs (voriconazole and natamycin) to treat Fusarium infections. RESULTS: We utilized combinations of chlorhexidine and natamycin or voriconazole against 20 clinical Fusarium strains in vitro using a checkerboard-based microdilution strategy. In order to more fully understand the synergistic interactions between voriconazole and chlorhexidine, we utilized a Galleria mellonella model to confirm the combined antifungal efficacy of chlorhexidine and voriconazole in vivo. We found that for voriconazole, natamycin, and chlorhexidine as single agents, the minimum inhibitory concentration (MIC) ranges were 2-8, 4-16, and > 16 µg/ml, respectively. In contrast, the MIC values for voriconazole and chlorhexidine were reduced to 0.25-1 and 1-2 µg/ml, respectively, when these agents were administered in combination, with synergy being observed for 90% of tested Fusarium strains. Combined chlorhexidine and natamycin treatment, in contrast, exhibited synergistic activity for only 10% of tested Fusarium strains. We observed no evidence of antagonism. Our in vivo model results further confirmed the synergistic antifungal activity of chlorhexidine and voriconazole. CONCLUSIONS: Our results offer novel evidence that voriconazole and chlorhexidine exhibit synergistic activity when used to suppress the growth of Fusarium spp., and these agents may thus offer value as a combination topical antifungal treatment strategy.

Choline Supplementation Sensitizes Legionella dumoffii to Galleria mellonella Apolipophorin III.

The growth of Legionella dumoffii can be inhibited by Galleria mellonella apolipophorin III (apoLp-III) which is an insect homologue of human apolipoprotein E., and choline-cultured L. dumoffii cells are considerably more susceptible to apoLp-III than bacteria grown without choline supplementation. In the present study, the interactions of apoLp-III with intact L. dumoffii cells cultured without and with exogenous choline were analyzed to explain the basis of this difference. Fluorescently labeled apoLp-III (FITC-apoLp-III) bound more efficiently to choline-grown L. dumoffii, as revealed by laser scanning confocal microscopy. The cell envelope of these bacteria was penetrated more deeply by FITC-apoLp-III, as demonstrated by fluorescence lifetime imaging microscopy analyses. The increased susceptibility of the choline-cultured L. dumoffii to apoLp-III was also accompanied by alterations in the cell surface topography and nanomechanical properties. A detailed analysis of the interaction of apoLp-III with components of the L. dumoffii cells was carried out using both purified lipopolysaccharide (LPS) and liposomes composed of L. dumoffii phospholipids and LPS. A single micelle of L. dumoffii LPS was formed from 12 to 29 monomeric LPS molecules and one L. dumoffii LPS micelle bound two molecules of apoLp-III. ApoLp-III exhibited the strongest interactions with liposomes with incorporated LPS formed of phospholipids isolated from bacteria cultured on exogenous choline. These results indicated that the differences in the phospholipid content in the cell membrane, especially PC, and LPS affected the interactions of apoLp-III with bacterial cells and suggested that these differences contributed to the increased susceptibility of the choline-cultured L. dumoffii to G. mellonella apoLp-III.

Miltefosine as an alternative strategy in the treatment of the emerging fungus Candida auris.

OBJECTIVES: Candida auris (C. auris) is an emerging fungal species that is able to develop multidrug resistance and outbreaks of invasive infections worldwide with high mortality rates. To increase the treatment options for C. auris infection this study assessed the efficacy of miltefosine (MFS), that has demonstrated a broad-spectrum antifungal action in vitro. This study aimed to: (i) evaluate the in vitro antifungal activity of MFS against C. auris clinical isolates in the planktonic and biofilm lifestyles; and (ii) compare the activity of MFS in its free form and encapsulated in alginate nanoparticles (MFS-AN) in Galleria mellonella larvae infected by C. auris. METHODS: The antifungal susceptibility test was performed using broth microdilution method and the in vivo treatment in Galleria mellonella larval infection model. RESULTS: MFS exhibited in vitro inhibitory effects at MICs ranging 1-4 µg/mL and fungicidal activity against planktonic cells of C. auris clinical isolates. MFS antibiofilm activity was observed during biofilm formation (0.25-4 µg/mL) and on pre-formed biofilms (16-32 µg/mL). Moreover, the dispersed cells from C. auris biofilms had a similar susceptibility to those obtained for planktonic cells. Treatment with free MFS or MFS-AN resulted in significant improvements in the survival and morbidity rates of Galleria mellonella larvae infected by C. auris. In addition, reduction of fungal burden (0.5-1 log CFU/g) and granuloma formation were observed when compared with the untreated group. CONCLUSIONS: The findings suggest that both the free MFS and MFS-AN have potential for the treatment of fungal infections caused by the emerging C. auris.

A novel biosafety level 2 compliant tuberculosis infection model using a ΔleuDΔpanCD double auxotroph of Mycobacterium tuberculosis H37Rv and Galleria mellonella.

Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialized animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output.

Non-walled spherical Acinetobacter baumannii is an important type of persister upon ß-lactam antibiotic treatment.

Bacterial persistence is one of the major causes of antibiotic treatment failure and the step stone for antibiotic resistance. However, the mechanism by which persisters arise has not been well understood. Maintaining a dormant state to prevent antibiotics from taking effect is believed to be the fundamental mechanistic basis, and persisters normally maintain an intact cellular structure. Here we examined the morphologies of persisters in Acinetobacter baumannii survived from the treatment by three major classes of antibiotics (i.e. ß-lactam, aminoglycoside, and fluoroquinolone) with microcopy and found that a fraction of enlarged spherical bacteria constitutes a major sub-population of bacterial survivors from ß-lactam antibiotic treatment, whereas survivors from the treatment of aminoglycoside and fluoroquinolone were less changed morphologically. Further studies showed that these spherical bacteria had completely lost their cell wall structures but could survive without any osmoprotective reagent. The spherical bacteria were not the viable-but-non-culturable cells and they could revive upon the removal of ß-lactam antibiotics. Importantly, these non-walled spherical bacteria also persisted during antibiotic therapy in vivo using Galleria mellonella as the infection model. Additionally, the combinational treatment on A. baumannii by ß-lactam and membrane-targeting antibiotic significantly enhanced the killing efficacy. Our results indicate that in addition to the dormant, structure intact persisters, the non-wall spherical bacterium is another important type of persister in A. baumannii. The finding suggests that targeting the bacterial cell membrane during ß-lactam chemotherapy could enhance therapeutic efficacy on A. baumannii infection, which might also help to reduce the resistance development of A. baumannii.

In Vitro and In Vivo Effects of the Polymyxin-Vorinostat Combination Therapy Against Multidrug-Resistant Gram-Negative Pathogens.

With the stagnancy of antibiotics development, polymyxins have become the last defense for treatment of multidrug-resistant (MDR) Gram-negative bacteria, whereas the effect of polymyxin monotherapy is limited by resistance. The objective of this study was to evaluate the effects of polymyxin B (PMNB)-vorinostat (SAHA) combination therapy against Gram-negative pathogens in vitro and in vivo. The antibacterial activities of PMNB and SAHA were evaluated by susceptibility testing. The synergistic effect was assessed by checkerboard tests and time-killing kinetics experiments. Cellular morphology studies and reactive oxygen species (ROS) assay were conducted to explore potential mechanisms. Also, Galleria mellonella models were made to evaluate the antibacterial effects in vivo. PMNB-SAHA had the synergistic effect against all tested isolates, reducing >2 log10 colony-forming units (CFU)/mL at 40 minutes, and showed more powerful antibacterial effects than PMNB alone in the 24-hour window. Cellular morphology study showed the change of membrane and disruption of integrity. ROS assay showed more oxidative stress in combination than PMNB or SAHA monotherapy. In animal models, PMNB-SAHA showed a higher survival rate than that of monotherapy. This study is the first to report the synergistic antibacterial effect of PMNB-SAHA therapy against MDR Gram-negative bacteria. Further clinical research is needed to confirm the results.

Midgut microbiota diversity of potato tuber moth associated with potato tissue consumed.

BACKGROUND: The potato tuber moth (PTM), Phthorimaea operculella (Zeller), is a worldwide pest that feeds on both the leaves and tubers of potato plants. PTM larvae can digest leaves, or tubers, resulting in serious damage to potato plants in the field and potato tubers in storage. To understand how midgut bacterial diversity is influenced by the consumption of these two tissue types, the symbiotic bacteria in the potato-feeding PTM midgut and the endophytic bacteria of potato tissues were analyzed. RESULTS: At the genus level, the bacterial community composition in the PTM midgut was influenced by the tissues consumed, owing to their different nutrient contents. Escherichia_Shigella and Enterobacter were the most dominant genera in the midgut of leaf-feeding and tuber-feeding PTMs, respectively. Interestingly, even though only present in low abundance in leaves and tubers, Escherichia_Shigella were dominantly distributed only in the midgut of leaf-feeding PTMs, indicating that specific accumulation of these genera have occurred by feeding on leaves. Moreover, Enterobacter, the most dominant genus in the midgut of tuber-feeding PTMs, was undetectable in all potato tissues, indicating it is gut-specific origin and tuber feeding-specific accumulation. Both Escherichia_Shigella and Enterobacter abundances were positively correlated with the dominant contents of potato leaves and tubers, respectively. CONCLUSIONS: Enrichment of specific PTM midgut bacterial communities was related to different nutrient levels in different tissues consumed by the insect, which in turn influenced host utilization. We provide evidence that a portion of the intestinal microbes of PTMs may be derived from potato endophytic bacteria and improve the understanding of the relationship between potato endophytic bacteria and the gut microbiota of PTMs, which may offer support for integrated management of this worldwide pest.

Vegetative development and host immune interaction of Ophiocordyceps sinensis within the hemocoel of the ghost moth larva, Thitarodes xiaojinensis.

Ophiocordyceps sinensis is an entomopathogenic fungus that infects ghost moth larva, forming the most valuable and rare traditional Chinese medicine, Chinese cordyceps. Our knowledge of the basic morphology and developmental biology of Chinese cordyceps is limited. In this study, morphological and ultrastructural observations of O. sinensis development in the hemocoel of Thitarodes xiaojinensis were obtained by multiple light and electron microscopy techniques, and the host immune reaction activities were determined. Our results indicated that fungal cells in the host hemocoel underwent morphotype transformations from blastospores to prehyphae to hyphae in sequence. The fusiform yeast-like blastospores were the initial cell type present in the host hemocoel and remained for 5 months or more; the encapsulation reaction and phenoloxidase activity of T. xiaojinensis hemolymph were inhibited during this period. When larvae entered the last instar, the blastospores switched to prehyphae and expanded throughout the host tissues, and then hyphae germinated from the prehyphae and mycelia formed, which finally led to host death. Considering the distinct differences between blastospores and hyphae, we identified prehyphae, which play important roles in fungal expansion, hyphae germination, and fusion formation among filaments. Notably, the elongation of prehyphae was strongly presumed to occur through fission but without separation of the two sister cells, in contrast to blastospore budding. During the morphotype transformation, the amount and composition of lipid droplets changed greatly, suggesting their important roles in these events. Overall, we provide a morphological and ultrastructural characterization of O. sinensis vegetative development within the hemocoel of T. xiaojinensis, identify and name the prehypha fungal cell type in entomopathogenic fungi for the first time, and conclude that O. sinensis infection causes sustained immunosuppression in T. xiaojinensis.

Tedizolid is a promising antimicrobial option for the treatment of Staphylococcus aureus infections in cystic fibrosis patients.

BACKGROUND: Tedizolid is a protein synthesis inhibitor in clinical use for the treatment of Gram-positive infections. Pulmonary MRSA infections are a growing problem in patients with cystic fibrosis (CF) and the efficacy of tedizolid-based therapy in CF pulmonary infections is unknown. OBJECTIVES: To evaluate the in vitro and in vivo activity of tedizolid and predict the likelihood of tedizolid resistance selection in CF-background Staphylococcus aureus strains. METHODS: A collection of 330 S. aureus strains (from adult and paediatric patients), either of normal or small colony variant (SCV) phenotypes, gathered at three CF centres in the USA was used. Tedizolid activity was assessed by broth microdilution, Etest and time-kill analysis. In vivo tedizolid efficacy was tested in a murine pneumonia model. Tedizolid in vitro mutants were obtained by 40 days of exposure and progressive passages. Whole genome sequencing of clinical S. aureus strains with reduced susceptibility to tedizolid was performed. RESULTS: MRSA strain MIC90s were tedizolid 0.12-0.25 mg/L and linezolid 1-2 mg/L; for MSSA strains, MIC90s were tedizolid 0.12 mg/L and linezolid 1-2 mg/L. Two strains, WIS 441 and Seattle 106, with tedizolid MICs of 2 mg/L and 1 mg/L, respectively, had MICs above the FDA tedizolid breakpoint (0.5 mg/L). Tedizolid at free serum concentrations exhibited a bacteriostatic effect. Mean bacterial burdens in lungs (log10 cfu/g) for WIS 423-infected mice were: control, 11.2±0.5; tedizolid-treated (10 mg/kg), 3.40±1.87; linezolid-treated (40 mg/kg), 4.51±2.1; and vancomycin-treated (30 mg/kg), 5.21±1.93. For WIS 441-infected mice the (log10 cfu/g) values were: control, 9.66±0.8; tedizolid-treated, 3.18±1.35; linezolid-treated 5.94±2.19; and vancomycin-treated, 4.35±1.7. CONCLUSIONS: These results suggest that tedizolid represents a promising therapeutic option for the treatment of CF-associated MRSA/MSSA infections, having potent in vivo activity and low resistance potential.