In Vitro Evaluation of Antimicrobial Peptides from the Black Soldier Fly (Hermetia Illucens) against a Selection of Human Pathogens.

Antimicrobial peptides (AMPs) are being explored as alternatives to traditional antibiotics to combat the rising antimicrobial resistance. Insects have proven to be a valuable source of new, potent AMPs with large structural diversity. For example, the black soldier fly has one of the largest AMP repertoires ever recorded in insects. Currently, however, this AMP collection has not yet undergone antimicrobial evaluation or in-depth in vitro characterization. This study evaluated the activity of a library of 36 black soldier fly AMPs against a panel of human pathogens (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus fumigatus) and a human cell line (MRC5-SV2). The activity profile of two cecropins (Hill-Cec1 and Hill-Cec10) with potent Gram-negative activity, was further explored by characterizing their hemolysis, time-to-kill kinetics, membrane-permeabilization properties, and anti-biofilm activity. Hill-Cec1 and Hill-Cec10 also showed high activity against other bacterial species, including Klebsiella pneumoniae and multi-drug resistant P. aeruginosa. Both AMPs are bactericidal and have a rapid onset of action with membrane-permeabilizing effects. Hill-Cec1 and Hill-Cec10 were also able to prevent P. aeruginosa biofilm formation, but no relevant effect was seen on biofilm eradication. Overall, Hill-Cec1 and Hill-Cec10 are promising leads for new antimicrobial development to treat critical infections caused by Gram-negative pathogens such as P. aeruginosa. IMPORTANCE With the ever growing antimicrobial resistance, finding new candidates for antimicrobial drug development is indispensable. Antimicrobial peptides have steadily gained attention as alternatives for conventional antibiotics, due to some highly desirable characteristics, such as their low propensity for resistance development. With this article, we aim to upgrade the knowledge on the activity of black soldier fly antimicrobial peptides and their potential as future therapeutics. To achieve this, we have evaluated for the first time a library of 36 synthetically produced peptides from the black soldier fly against a range of human pathogens and a human cell line. Two selected peptides have undergone additional testing to characterize their antimicrobial profile against P. aeruginosa, a clinically important Gram-negative pathogen with a high established resistance. Overall, this research has contributed to the search for new peptide drug leads to combat the rising antimicrobial resistance.

Culture-Independent and Culture-Dependent Characterization of the Black Soldier Fly Gut Microbiome Reveals a Large Proportion of Culturable Bacteria with Potential for Industrial Applications.

Black soldier fly larvae (BSFL) are fast-growing, resilient insects that can break down a variety of organic substrates and convert them into valuable proteins and lipids for applications in the feed industry. Decomposition is mediated by an abundant and versatile gut microbiome, which has been studied for more than a decade. However, little is known about the phylogeny, properties and functions of bacterial isolates from the BSFL gut. We therefore characterized the BSFL gut microbiome in detail, evaluating bacterial diversity by culture-dependent methods and amplicon sequencing of the 16S rRNA gene. Redundant strains were identified by genomic fingerprinting and 105 non-redundant isolates were then tested for their ability to inhibit pathogens. We cultivated representatives of 26 genera, covering 47% of the families and 33% of the genera detected by amplicon sequencing. Among these isolates, we found several representatives of the most abundant genera: Morganella, Enterococcus, Proteus and Providencia. We also isolated diverse members of the less-abundant phylum Actinobacteria, and a novel genus of the order Clostridiales. We found that 15 of the isolates inhibited at least one of the tested pathogens, suggesting a role in helping to prevent colonization by pathogens in the gut. The resulting culture collection of unique BSFL gut bacteria provides a promising resource for multiple industrial applications.

Cottonseed Press Cake as a Potential Diet for Industrially Farmed Black Soldier Fly Larvae Triggers Adaptations of Their Bacterial and Fungal Gut Microbiota.

Black soldier fly larvae (Hermetia illucens, Diptera: Stratiomyidae) are used for the bioconversion of organic side products into valuable compounds such as proteins, lipids and chitin. However, the economic competitiveness of farmed insects compared to conventional protein production systems in agriculture and aquaculture depends on the availability of large quantities of inexpensive insect feed. Cottonseed press cake (CPC) is a side-stream of cotton production that is rich in proteins and lipids but unsuitable as feed for several farmed animals, except ruminants, due to the presence of the anti-nutritional sesquiterpenoid gossypol. Here, we tested CPC as a feed for black soldier fly larvae and studied the impact of this diet on the gut microbiome. Larvae reared on CPC developed normally and even showed a shorter life-cycle, but were smaller at the end of larval development than control larvae reared on chicken feed. The adaptability of the larvae to different diets is mediated by their versatile gut microbiome, which facilitates digestion and detoxification. We therefore used amplicon sequencing to analyze the bacterial and fungal communities associated with larvae reared on each diet, revealing differences between the larval guts and frass (residual feed substrate) as well as differences between the two diet groups. For example, Actinomycetaceae and Aspergillaceae were significantly enriched in guts of the CPC diet group and may help to metabolize compounds such as gossypol. Potentially probiotic yeasts and beneficial Enterobacteriaceae, which presumably belong to the core microbiota, were detected in high relative abundance in the gut and frass, indicating a functional role of these microbes, especially the protection against pathogens. We conclude that CPC may be suitable as an inexpensive and environmentally sustainable feed for the industrial rearing of black soldier flies.

Ereboglobus luteus gen. nov. sp. nov. from cockroach guts, and new insights into the oxygen relationship of the genera Opitutus and Didymococcus (Verrucomicrobia: Opitutaceae).

We isolated a novel member of the phylum Verrucomicrobia from the hindgut of the cockroach Shelfordella lateralis. Strain Ho45 is a yellow-pigmented, motile coccus that represents a new genus-level lineage with less than 93% sequence similarity to the 16S rRNA genes of other species in the family Opitutaceae. Ultrastructural analysis revealed a Gram-negative cell envelope with an outer membrane and a periplasmic space. In its ability to ferment sugars to propionate and acetate as major products, strain Ho45 resembles its closest relative, Opitutus terrae. However, the strains differed in their relationship to oxygen. Although strain Ho45 grew and consumed oxygen at sub-atmospheric concentrations (1-4%), both growth rate and cell yield decreased strongly with increasing oxygen concentration in the headspace. By contrast, O. terrae, previously described as an obligate anaerobe, proved to be facultatively aerobic, with highest growth rates and cell yields at 2% and 16% oxygen, respectively. Also the closely related Didymococcus (Diplosphaera) colitermitum, previously described as an obligately aerobic microaerophile, showed a fermentative metabolism under anoxic conditions, forming the same products from glucose as strain Ho45 and O. terrae. Based on phenotypic and phylogenetic evidence, we propose strain Ho45 as the type strain of a novel genus, Ereboglobus luteus gen. nov. sp. nov., and provide an emended description of the family Opitutaceae and the genera Opitutus and Didymococcus.

Breznakia blatticola gen. nov. sp. nov. and Breznakia pachnodae sp. nov., two fermenting bacteria isolated from insect guts, and emended description of the family Erysipelotrichaceae.

Two novel, obligately anaerobic Firmicutes from the family Erysipelotrichaceae were isolated from the intestinal tracts of a cockroach (strain ErySL, Shelfordella lateralis) and a scarab beetle larva (strain Pei061, Pachnoda ephippiata). Phylogenetic analysis indicated that the strains belong to a monophyletic group of hitherto uncultured bacteria from insect guts that are only distantly related to any described species (<90% 16S rRNA gene sequence similarity). Ultrastructural analysis revealed a Gram-positive cell envelope and, in the case of strain ErySL, a wide electron-lucent space between the cytoplasmic membrane and cell wall. In older cultures, cells formed pleomorphic rods with a thicker peptidoglycan layer. Both strains were obligately anaerobic and fermented glucose to formate, ethanol, and acetate as major products, but strain Pei061 tolerated up to 1% oxygen in the headspace. The same type of metabolism was observed with Erysipelothrix inopinata, except that the latter grew, albeit poorly, even under air. However, previous claims of a microaerophilic or facultatively anaerobic metabolism in the genus Erysipelothrix could not be substantiated. Based on phenotypic and phylogenetic evidence, we propose to classify the isolates as members of a new genus, Breznakia blatticola gen. nov. sp. nov. and Breznakia pachnodae sp. nov., with strain ErySL(T) (=DSM 28867(T)=JCM 30190(T)) and strain Pei061(T) (=DSM 16784(T)=JCM 30191(T)) as type strains, and provide an emended description of the family Erysipelotrichaceae.

Oxygen Affects Gut Bacterial Colonization and Metabolic Activities in a Gnotobiotic Cockroach Model.

The gut microbiota of termites and cockroaches represents complex metabolic networks of many diverse microbial populations. The distinct microenvironmental conditions within the gut and possible interactions among the microorganisms make it essential to investigate how far the metabolic properties of pure cultures reflect their activities in their natural environment. We established the cockroach Shelfordella lateralis as a gnotobiotic model and inoculated germfree nymphs with two bacterial strains isolated from the guts of conventional cockroaches. Fluorescence microscopy revealed that both strains specifically colonized the germfree hindgut. In diassociated cockroaches, the facultatively anaerobic strain EbSL (a new species of Enterobacteriaceae) always outnumbered the obligately anaerobic strain FuSL (a close relative of Fusobacterium varium), irrespective of the sequence of inoculation, which showed that precolonization by facultatively anaerobic bacteria does not necessarily favor colonization by obligate anaerobes. Comparison of the fermentation products of the cultures formed in vitro with those accumulated in situ indicated that the gut environment strongly affected the metabolic activities of both strains. The pure cultures formed the typical products of mixed-acid or butyrate fermentation, whereas the guts of gnotobiotic cockroaches accumulated mostly lactate and acetate. Similar shifts toward more-oxidized products were observed when the pure cultures were exposed to oxygen, which corroborated the strong effects of oxygen on the metabolic fluxes previously observed in termite guts. Oxygen microsensor profiles of the guts of germfree, gnotobiotic, and conventional cockroaches indicated that both gut tissue and microbiota contribute to oxygen consumption and suggest that the oxygen status influences the colonization success.