Bacteriophage cocktail shows no toxicity and improves the survival of Galleria mellonella infected with Klebsiella spp.

Klebsiella spp. are causative agents of healthcare-associated infections in patients who are immunocompromised and use medical devices. The antibiotic resistance crisis has led to an increase in infections caused by these bacteria, which can develop into potentially life-threatening illnesses if not treated swiftly and effectively. Thus, new treatment options for Klebsiella are urgently required. Phage therapy can offer an alternative to ineffective antibiotic treatments for antibiotic-resistant bacteria infections. The aim of the present study was to produce a safe and effective phage cocktail treatment against Klebsiella pneumoniae and Klebsiella oxytoca, both in liquid in vitro culture and an in vivo Galleria mellonella infection model. The phage cocktail was significantly more effective at killing K. pneumoniae and K. oxytoca strains compared with monophage treatments. Preliminary phage cocktail safety was demonstrated through application in the in vivo G. mellonella model: where the phage cocktail induced no toxic side effects in G. mellonella. In addition, the phage cocktail significantly improved the survival of G. mellonella when administered as a prophylactic treatment, compared with controls. In conclusion, our phage cocktail was demonstrated to be safe and effective against Klebsiella spp. in the G. mellonella infection model. This provides a strong case for future treatment for Klebsiella infections, either as an alternative or adjunct to antibiotics.IMPORTANCEKlebsiella infections are a concern in individuals who are immunocompromised and are becoming increasingly difficult to treat with antibiotics due to their drug-resistant properties. Bacteriophage is one potential alternative therapy that could be used to tackle these infections. The present study describes the design of a non-toxic phage cocktail that improved the survival of Galleria mellonella infected with Klebsiella. This phage cocktail demonstrates potential for the safe and effective treatment of Klebsiella infections, as an adjunct or alternative to antibiotics.

Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model.

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.

Analysis of the virulence, infection process, and extracellular enzyme activities of Aspergillus nomius against the Asian corn borer, Ostrinia furnacalis guenée (Lepidoptera: Crambidae).

The control of Ostrinia furnacalis, a major pest of maize in Xinjiang, is challenging owing to the occurrence of resistant individuals. Entomopathogenic fungi (EPF) are natural insect regulators used as substitutes for synthetic chemical insecticides. The fungus Aspergillus nomius is highly pathogenic to O. furnacalis; however, its virulence characteristics have not been identified. This study aimed to analyse the lethal efficacy, mode of infection on the cuticle, and extracellular enzyme activity of A. nomius against O. furnacalis. We found that the mortality and mycosis of O. furnacalis were dose-dependent when exposed to A. nomius and varied at different life stages. The egg-hatching and adult emergence rates decreased with an increase in conidial suspension. The highest mortality (83.33%, 7 d post-infection [DPI]) and mycosis (74.33%, 7 DPI) and the lowest mortality response (8.52 × 103 conidia mL-1) and median lethal time (4.91 d) occurred in the 3rd instar larvae of O. furnacalis. Scanning electron microscopy indicated that numerous conidia germination and infection structure formation may have contributed to the high pathogenicity of A. nomius against O. furnacalis. There were significant correlations between O. furnacalis mortality and the activities of extracellular protease, lipase, and chitinase of A. nomius. This study revealed the infection process of the highly pathogenic A. nomius against O. furnacalis, providing a theoretical basis and reference for strain improvement and field application of EPF.

Synergistic Effects of Combining Three Commercial Bioproducts Against Tuta absoluta (Meyrick) Larvae (Lepidoptera: Gelechiidae).

<b>Background and Objective:</b> The use of entomopathogenic agents for crop pest management is a viable alternative to synthetic chemical pesticides. <i>Beauveria bassiana</i> (Bals.) and <i>Metarhizium anisopliae</i> (Metsch.) are fungi considered the most promising extensively widely applied bio-control agents in protecting a wide range of economic crops. Fungal toxins are thought to play a crucial part in the pathogenicity process during insect infestation. The bioinsecticides' synergy could help to control the invasive pest more safely and effectively. <b>Materials and Methods:</b> Suspensions of Beauveroz (<i>Beauveria</i> <i>bassiana</i>) and Metarhoz-P (<i>Metarhizium anisopliae</i>), were evaluated as to their virulence against <i>T.</i> <i>absoluta</i> larvae at 3 different doses. As a comparison, Abamectin was utilized as a positive control, while water was used as a negative control. <b>Results:</b> All the commercial compounds caused significant mortality among <i>T.</i> <i>absoluta</i> larvae, with approximately 52% mortality after 5 days of the treatment. Over 5 days, mortality of <i>T.</i> <i>absoluta</i> larvae when exposed to a combined treatment of <i>B.</i> <i>bassiana</i>, <i>M.</i> <i>anisopliae</i> and Abamectin reached 92%. The results under field conditions, showed significant differences (p<0.001) among these products while adding the surfactants increased the mortality larvae. Combined treatments of these 3 commercial compounds showed a synergistic effect acceded the effect obtained using each compound alone. Bio-pesticides, <i>B.</i> <i>bassiana</i> and <i>M.</i> <i>anisopliae</i> formulations caused mortality rates among <i>T.</i> <i>absoluta</i> larvae similar to the Abamectin treatment. <b>Conclusion:</b> Observations indicated that both fungus candidates and Abamectin proved effective against <i>T. absoluta</i> larvae. The combined use showed a high potentiality indicating a positive synergistic effect.

Fungi are more transient than bacteria in caterpillar gut microbiomes.

Despite an increasing number of studies on caterpillar (Insecta: Lepidoptera) gut microbiota, bacteria have been emphasized more than fungi. Therefore, we lack data on whether fungal microbiota is resident or transient and shaped by factors similar to those of bacteria. We sampled nine polyphagous caterpillar species from several tree species at multiple sites to determine the factors shaping leaf and gut bacterial and fungal microbiota as well as the extent to which caterpillars acquire microbiota from their diet. We performed 16S and ITS2 DNA metabarcoding of the leaves and guts to determine the composition and richness of the respective microbiota. While spatial variables shaped the bacterial and fungal microbiota of the leaves, they only affected fungi in the guts, whereas the bacteria were shaped primarily by caterpillar species, with some species harboring more specific bacterial consortia. Leaf and gut microbiota significantly differed; in bacteria, this difference was more pronounced. The quantitative similarity between leaves and guts significantly differed among caterpillar species in bacteria but not fungi, suggesting that some species have more transient bacterial microbiota. Our results suggest the complexity of the factors shaping the gut microbiota, while highlighting interspecific differences in microbiota residency within the same insect functional group.

Fungal consortium of two Beauveria bassiana strains increases their virulence, growth, and resistance to stress: A metabolomic approach.

The use of two or more microorganisms in a microbial consortium has been increasingly applied in the biological control of diseases and pests. Beauveria bassiana is one of the most widely studied fungal species in biological control, yet little is known about its role in fungal consortiums. In a previous study, our group found that a consortium formed by two strains of B. bassiana had significantly greater biocontrol potential against the polyphagous caterpillars Duponchelia fovealis (Lepidoptera: Crambidae) than either strain on its own. In this study, we use GC-MS and LC-MS/MS to evaluate and discuss the metabolomics of the consortium. A total of 21 consortium biomarkers were identified, corresponding to 14 detected by LC-MS/MS and seven by GC-MS. Antioxidant and anti-inflammatory mechanisms are the main properties of the metabolites produced by the consortium. These metabolites can depress the insect's immune system, increasing its vulnerability and, hence, the fungal virulence of the consortium. In light of these results, we propose an action model of insect mortality due to the metabolites secreted by the consortium. The model includes the inhibition of defense mechanisms such as pro-inflammatory interleukin secretion, cell migration, cell aggregation, Dif, Dorsal and Relish gene transcription, and JAK/STAT and JNK signaling pathways. It also promotes the cleaning of oxidative molecules, like ROS, NOS, and H2O2, and the induction of virulence factors.

Octanoic Acid-An Insecticidal Metabolite of Conidiobolus coronatus (Entomopthorales) That Affects Two Majors Antifungal Protection Systems in Galleria mellonella (Lepidoptera): Cuticular Lipids and Hemocytes.

The food flavour additive octanoic acid (C8:0) is also a metabolite of the entomopathogenic fungus Conidiobolus coronatus, which efficiently infects and rapidly kills Galleria mellonella. GC-MS analysis confirmed the presence of C8:0 in insecticidal fraction FR3 extracted from C. coronatus filtrate. Topical administration of C8:0 had a dose-dependent effect on survival rates of larvae but not on pupation or adult eclosion times of the survivors. Topically applied C8:0 was more toxic to adults than larvae (LD100 for adults 18.33 ± 2.49 vs. 33.56 ± 2.57 µg/mg of body mass for larvae). The administration of C8:0 on the cuticle of larvae and adults, in amounts corresponding to their LD50 and LD100 doses, had a considerable impact on the two main defense systems engaged in protecting against pathogens, causing serious changes in the developmental-stage-specific profiles of free fatty acids (FFAs) covering the cuticle of larvae and adults and damaging larval hemocytes. In vitro cultures of G. mellonella hemocytes, either directly treated with C8:0 or taken from C8:0 treated larvae, revealed deformation of hemocytes, disordered networking, late apoptosis, and necrosis, as well as caspase 1-9 activation and elevation of 8-OHdG level. C8:0 was also confirmed to have a cytotoxic effect on the SF-9 insect cell line, as determined by WST-1 and LDH tests.

Thermal Requirements and Life Table Parameters of Tomato Leaf Miner Tuta absoluta (Lepidoptera: Gelechiidae) in Egypt.

<b>Background and Objective:</b> The tomato leaf miner, <i>Tuta absoluta</i> (Meyrick) is being a serious pest to tomato cultivations in Egypt since 2009. The present study was carried out to calculate the developmental parameters of insects based on temperature degree. <b>Materials and Methods:</b> The influence of 3 tested temperatures (20, 24, 28°C) were examined to evaluate its effect on the developmental stages of <i>T. absoluta</i>. Developmental thresholds and needed heat units for insect stages were mathematically calculated according to developmental rates. <b>Results:</b> Developmental threshold for egg stage and mean thermal units were calculated to be 7°C and 86.2 DD's. The developmental threshold for the larval stage was 10°C, while mean thermal units were calculated to be 310.8 DD's. Percentages mortality of larval stage were 52, 74, 74 and 100% at 20, 24, 28 and 32°C, respectively. For the pupal stage developmental threshold and mean thermal units required for completing the pupal stage was 11.2°C and 132.2 DD's. For an adult, zero of the developmental threshold female and of male were 11.2 and 9.8°C, respectively. The mean required heat units for female and male was 142.3 and 136.7 DD's Life table parameters such as net Reproduction Rate (R_◦), Mean Generation Time (Gt), Intrinsic Rate of Increase (r_m), Finite Rate of Increase (λ) and Population Double Time (Dt) were calculated at three tested temperatures. <b>Conclusion:</b> Estimating thermal heat units of <i>T. absoluta</i> help in predicting the field generations of the insect and improve planning the integrated pest management.

A Short-Type Peptidoglycan Recognition Protein 1 (PGRP1) Is Involved in the Immune Response in Asian Corn Borer, Ostrinia furnacalis (Guenée).

The insect immune response is initiated by the recognition of invading microorganisms. Peptidoglycan recognition proteins (PGRPs) function primarily as pattern recognition receptors by specifically binding to peptidoglycans expressed on microbial surfaces. We cloned a full-length cDNA for a PGRP from the Asian corn borer Ostrinia furnacalis (Guenée) and designated it as PGRP1. PGRP1 mRNA was mainly detected in the fat bodies and hemocytes. Its transcript levels increased significantly upon bacterial and fungal challenges. Purified recombinant PGRP1 exhibited binding activity to the gram-positive Micrococcus luteus, gram-negative Escherichia coli, entomopathogenic fungi Beauveria bassiana, and yeast Pichia pastoris. The binding further induced their agglutination. Additionally, PGRP1 preferred to bind to Lys-type peptidoglycans rather than DAP-type peptidoglycans. The addition of recombinant PGRP1 to O. furnacalis plasma resulted in a significant increase in phenoloxidase activity. The injection of recombinant PGRP1 into larvae led to a significantly increased expression of several antimicrobial peptide genes. Taken together, our results suggest that O. furnacalis PGRP1 potentially recognizes the invading microbes and is involved in the immune response in O. furnacalis.

Polyurethane foam as an inert support using concentrated media improves quality and spore production from Bacillus thuringiensis.

Bacillus thuringiensis (Bt) (Bacillales:Bacillaceae) is a gram-positive bacterium that produces spores, several virulence factors and insecticidal toxins, making this microorganism the most used biopesticide worldwide. The use of inert supports such as polyurethane foam (PUF) in solid cultures has been a great alternative to produce various metabolites, including those produced by Bt. In this study we compared the yields, productivity and quality of the spores by two wild strains of Bt, (Y15 and EA3), grown in media with high substrate concentration in both culture systems: liquid and solid (PUF as solid inert support). Both strains showed 2.5- to 30-fold increases in spore production and productivity in solid culture, which showed an even greater increase when considering the spores retained in the PUF observed by scanning electron microscopy. Moreover, spore produced in solid culture showed up to sevenfold higher survival after a heat-shock treatment, relative to spores from liquid culture. The infectivity against larvae of Galleria mellonella (Lepidoptera:Pyralidae) improved also in spores from solid cultures. This comparison showed that the culture of Bt on solid support has clear advantages over liquid culture in terms of the production and quality of spores, and that those advantages can be attributed only to the culture system, as the same media composition was used in both systems.

Comparison of Gut Bacterial Communities of Grapholita molesta (Lepidoptera: Tortricidae) Reared on Different Host Plants.

Intestinal symbiotic bacteria have played an important role in the digestion, immunity detoxification, mating, and reproduction of insects during long-term coevolution. The oriental fruit moth, Grapholita molesta, is an important fruit tree pest worldwide. However, the composition of the G. molesta microbial community, especially of the gut microbiome, remains unclear. To explore the differences of gut microbiota of G. molesta when reared on different host plants, we determined the gut bacterial structure when G. molesta was transferred from an artificial diet to different host plants (apples, peaches, nectarines, crisp pears, plums, peach shoots) by amplicon sequencing technology. The results showed that Proteobacteria and Firmicutes are dominant in the gut microbiota of G. molesta. Plum-feeding G. molesta had the highest richness and diversity of gut microbiota, while apple-feeding G. molesta had the lowest. PCoA and PERMANOVA analysis revealed that there were significant differences in the gut microbiota structure of G. molesta on different diets. PICRUSt2 analysis indicated that most of the functional prediction pathways were concentrated in metabolic and cellular processes. Our results confirmed that gut bacterial communities of G. molesta can be influenced by host diets and may play an important role in host adaptation.

Hyperexpression of CDRs and HWP1 genes negatively impacts on Candida albicans virulence.

C. albicans is a commensal organism present in the human microbiome of more than 60% of the healthy population. Transition from commensalism to invasive candidiasis may occur after a local or a general failure of host's immune system. This transition to a more virulent phenotype may reside either on the capacity to form hyphae or on an acquired resistance to antifungal drugs. Indeed, overexpression of genes coding drug efflux pumps or adhesins, cell wall proteins facilitating the contact between the fungus and the host, usually marks the virulence profile of invasive Candida spp. In this paper, we compare virulence of two clinical isolates of C. albicans with that of laboratory-induced resistant strains by challenging G. mellonella larvae with these pathogens along with monitoring transcriptional profiles of drug efflux pumps genes CDR1, CDR2, MDR1 and the adhesin genes ALS1 and HWP1. Although both clinical isolates were found resistant to both fluconazole and micafungin they were found less virulent than laboratory-induced resistant strains. An unexpected behavior emerged for the former clinical isolate in which three genes, CDR1, CDR2 and HWP1, usually correlated with virulence, although hyperexpressed, conferred a less aggressive phenotype. On the contrary, in the other isolate, we observed a decreased expression of CDR1, CDR2 and HWP1as well as of MDR1 and ALS1 that may be consistent with the less aggressive performance observed in this strain. These altered gene expressions might directly influence Candida virulence or they might be an epiphenomenon of a vaster rearrangement occurred in these strains during the challenge with the host's environment. An in-deepth comprehension of this scenario could be crucial for developing interventions able to counteract C. albicans invasiveness and lethality.

Infected insect gut reveals differentially expressed proteins for cellular redox, metal resistance and secretion system in Yersinia enterocolitica-Helicoverpa armigera pathogenic model.

OBJECTIVE: Mouse infection models are frequently used to study the host-pathogen interaction studies. However, due to several constraints, there is an urgent need for a simple, rapid, easy to handle, inexpensive, and ethically acceptable in vivo model system for studying the virulence of enteropathogens. Thus, the present study was performed to develop the larvae of Helicoverpa armigera as a rapid-inexpensive in vivo model system to evaluate the effect of Yersinia enterocolitica strain 8081 on its midgut via a label-free proteomic approach. RESULTS: Helicoverpa armigera larvae fed with Yersinia enterocolitica strain 8081 manifested significant reduction in body weight and damage in midgut. On performing label-free proteomic study, secretory systems, putative hemolysin, and two-component system emerged as the main pathogenic proteins. Further, proteome comparison between control and Yersinia added diet-fed (YADF) insects revealed altered cytoskeletal proteins in response to increased melanization (via a prophenoloxidase cascade) and free radical generation. In concurrence, FTIR-spectroscopy, and histopathological and biochemical analysis confirmed gut damage in YADF insects. Finally, the proteome data suggests that the mechanism of infection and the host response in Y. enterocolitica-H. armigera system mimics Yersinia-mammalian gut interactions. CONCLUSIONS: All data from current study collectively suggest that H. armigera larva can be considered as a potential in vivo model system for studying the enteropathogenic infection by Y. enterocolitica strain 8081.

Newly Isolated Animal Pathogen Corynebacterium silvaticum Is Cytotoxic to Human Epithelial Cells.

Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.

Molecular Evidence of Rickettsia felis in Phereoeca sp.

Rickettsia felis is an obligate intracellular bacterium capable of infecting ticks, fleas, lice, and other arthropods. This bacterium is classified as a member of the Transitional Group (TRG) Rickettsia. It is known the evidence of R. felis mutualistic and obligatory relationship with some eukaryote organisms. However, there aren't scientific accounts of R. felis and moths of the order Lepidoptera association. The current work reports the first identification of the bacteria R. felis in Phereoeca sp. For that, a polymerase chain reaction (PCR) assay using gltA, ompA, and ompB genes was used. The nucleotide sequences showed 100% of identity with other Rickettsia felis sequences. The genus-level identification of the moth larvae was performed by morphological taxonomic keys and PCR analysis of the cytochrome oxidase I (COI) gene. The nucleotide sequenced showed 94.94% similarity with the species Phereoeca praecox. However, with the low number of sequences deposited in the databases, the species was classified as Phereoeca sp. The results suggest that R. felis may develop in an organism without blood-feeding behavior (Lepidoptera), as it has been demonstrated for booklice (Psocoptera). Further investigation is necessary in order to confirm pathogenic or mutualistic association with moths.

Management alternatives for Carmenta theobromae (Busck, 1910) (Lepidoptera: Sesiidae) and Simplicivalva ampliophilobia (Lepidoptera: Cossidae), limiting pests of guava in Colombia.

The larval stages of Carmenta theobromae Busck (1910) and Simplicivalva ampliophilobia Davis, Gentili-Poole and Mitter (2008) attack the subcortical zone and pith in guava trees, respectively, in the first productive nucleus of fruit trees in Colombia: Hoya del Río Suárez (HRS). The presence of pest insects has been reported in 98% of the farms sampled in HRS (n = 124), with up to 96 and 11 simultaneous larvae per tree, respectively. Although the aspects of the basic biology and life cycle of both pests have been resolved, there are no strategies for managing populations in the field. Therefore, the aim of this study was to evaluate different management alternatives under laboratory and field conditions in HRS. In laboratory conditions, a completely randomized design was used in two separate experiments, each with six treatments: T1: Spinosad (a mixture of Spinosad A and D); T2: S-1,2-di(ethoxycarbonyl) ethyl 0,0-dimethylphosphorodithioate (chemical control); T3: Lecanicillium lecanii; T4: Beauveria bassiana; T5: Mix of B. bassiana and B. brongniartii, and T6: distilled water (control). The number of dead larvae per replicate per treatment was evaluated (DL), with experimental units of five and three larvae, respectively. In the field, to the two best alternatives found for each pest in the laboratory, pruning and keeping the area around the plants free of weeds were added as cultural management, in two separate additional experiments, each with three larvae as experimental unit per treatment. For C. theobromae, the best laboratory alternatives were chemical control (DL: 3.78) and L. lecanii (DL: 2.33), followed without statistical differences by B. bassiana (DL: 1.67). In the field, the virulence of B. bassiana improved (DL: 3), and together with pruning and keeping the area around the plants clear of weeds (DL: 3), they stood out as the best alternatives. For S. ampliophilobia under laboratory conditions, the best alternatives were Spinosad (2.74) and chemical control (DL: 2.66), without significant difference. In the field, there were no statistical differences between the alternatives, except for the control. This statistical parity of cultural practices, and biological and chemical management is an argument in favor of the use of the former to the detriment of the third, especially when the harmful effects of the molecule S-1,2 di (ethoxycarbonyl) ethyl 0, 0-dimethyl phosphorodithioate have been proven in air, water and agricultural soils, in addition to its association with thyroid cancer in humans. This is a strong argument to favor the use of synergies of cultural and biological management methods framed in IPM, as opposed to the use of chemical agents whose harmful effects are strongly documented, and whose use is becoming increasingly prohibited.

Optimising efficacy of antibiotics against systemic infection by varying dosage quantities and times.

Mass production and use of antibiotics has led to the rise of resistant bacteria, a problem possibly exacerbated by inappropriate and non-optimal application. Antibiotic treatment often follows fixed-dose regimens, with a standard dose of antibiotic administered equally spaced in time. But are such fixed-dose regimens optimal or can alternative regimens be designed to increase efficacy? Yet, few mathematical models have aimed to identify optimal treatments based on biological data of infections inside a living host. In addition, assumptions to make the mathematical models analytically tractable limit the search space of possible treatment regimens (e.g. to fixed-dose treatments). Here, we aimed to address these limitations by using experiments in a Galleria mellonella (insect) model of bacterial infection to create a fully parametrised mathematical model of a systemic Vibrio infection. We successfully validated this model with biological experiments, including treatments unseen by the mathematical model. Then, by applying artificial intelligence, this model was used to determine optimal antibiotic dosage regimens to treat the host to maximise survival while minimising total antibiotic used. As expected, host survival increased as total quantity of antibiotic applied during the course of treatment increased. However, many of the optimal regimens tended to follow a large initial 'loading' dose followed by doses of incremental reductions in antibiotic quantity (dose 'tapering'). Moreover, application of the entire antibiotic in a single dose at the start of treatment was never optimal, except when the total quantity of antibiotic was very low. Importantly, the range of optimal regimens identified was broad enough to allow the antibiotic prescriber to choose a regimen based on additional criteria or preferences. Our findings demonstrate the utility of an insect host to model antibiotic therapies in vivo and the approach lays a foundation for future regimen optimisation for patient and societal benefits.

Human antimicrobial peptide, LL-37, induces non-inheritable reduced susceptibility to vancomycin in Staphylococcus aureus.

Antimicrobial peptides (AMPs) are central components of the innate immune system providing protection against pathogens. Yet, serum and tissue concentrations vary between individuals and with disease conditions. We demonstrate that the human AMP LL-37 lowers the susceptibility to vancomycin in the community-associated methicillin-resistant S. aureus (CA-MRSA) strain FPR3757 (USA300). Vancomycin is used to treat serious MRSA infections, but treatment failures occur despite MRSA strains being tested susceptible according to standard susceptibility methods. Exposure to physiologically relevant concentrations of LL-37 increased the minimum inhibitory concentration (MIC) of S. aureus towards vancomycin by 75%, and resulted in shortened lag-phase and increased colony formation at sub-inhibitory concentrations of vancomycin. Computer simulations using a mathematical antibiotic treatment model indicated that a small increase in MIC might decrease the efficacy of vancomycin in clearing a S. aureus infection. This prediction was supported in a Galleria mellonella infection model, where exposure of S. aureus to LL-37 abolished the antimicrobial effect of vancomycin. Thus, physiological relevant concentrations of LL-37 reduce susceptibility to vancomycin, indicating that tissue and host specific variations in LL-37 concentrations may influence vancomycin susceptibility in vivo.

The synergistic antifungal effects of gypenosides combined with fluconazole against resistant Candida albicans via inhibiting the drug efflux and biofilm formation.

The increased resistance of Candida to conventional antifungals brings great challenges for the clinical treatment of Candida infections. Recently, more attention has been paid to the research on combination therapy, which is a potential therapeutic approach for overcoming Candida resistance. In the present study, we first investigated the interaction between gypenosides (Gyp) and fluconazole (FLC) against Candida albicans (C. albicans) in vitro and in vivo. The in vitro test revealed a synergistic antifungal activity between Gyp and FLC against FLC-resistant (FLCR) C. albicans and indifferent effects for FLC-susceptible (FLCS) C. albicans, with the fractional inhibitory concentration index of 0.2539-0.2578 and 1-1.5, respectively. Besides, Gyp displayed synergistic interaction with FLC against FLCRC. albicans performed biofilm over 4 h, with the fractional inhibitory concentration index <0.5. In vivo, the combined antifungal efficacy of Gyp with FLC was evaluated by Galleria mellonella (G. mellonella) larvae. Gyp plus FLC prolonged the survival rate and reduced tissue invasion of larvae infected with FLCRC. albicans. Further experiments to get a first hint at what antifungal mechanisms might be inhibition of early biofilm formation, suppression of drug efflux, and inhibition of yeast-hyphal conversion. These findings will provide a new approach for the treatment of C. albicans infection.

Antibiotics Act with vB_AbaP_AGC01 Phage against Acinetobacter baumannii in Human Heat-Inactivated Plasma Blood and Galleria mellonella Models.

Increasing multidrug resistance has led to renewed interest in phage-based therapy. A combination of the bacteriophages and antibiotics presents a promising approach enhancing the phage therapy effectiveness. First, phage candidates for therapy should be deeply characterized. Here we characterize the bacteriophage vB_AbaP_AGC01 that poses antibacterial activity against clinical Acinetobacter baumannii strains. Moreover, besides genomic and phenotypic analysis our study aims to analyze phage-antibiotic combination effectiveness with the use of ex vivo and in vivo models. The phage AGC01 efficiently adsorbs to A. baumannii cells and possesses a bacteriolytic lifecycle resulting in high production of progeny phages (317 ± 20 PFU × cell-1). The broad host range (50.27%, 93 out of 185 strains) against A. baumannii isolates and the inability of AGC01 to infect other bacterial species show its high specificity. Genomic analysis revealed a high similarity of the AGC01 genome sequence with that of the Friunavirus genus from a subfamily of Autographivirinae. The AGC01 is able to significantly reduce the A. baumannii cell count in a human heat-inactivated plasma blood model (HIP-B), both alone and in combination with antibiotics (gentamicin (GEN), ciprofloxacin (CIP), and meropenem (MER)). The synergistic action was observed when a combination of phage treatment with CIP or MER was used. The antimicrobial activity of AGC01 and phage-antibiotic combinations was confirmed using an in vivo larva model. This study shows the greatest increase in survival of G. mellonella larvae when the combination of phage (MOI = 1) and MER was used, which increased larval survival from 35% to 77%. Hence, AGC01 represents a novel candidate for phage therapy. Additionally, our study suggests that phages and antibiotics can act synergistically for greater antimicrobial effect when used as combination therapy.