Spores, Bacterial

The performance of the overall workflow or of its individual modules was assessed using a spectrum of different matrices that can be grouped into the five categories liquids, faeces, tissue, vectors, and food. The processed samples were mostly diagnostic specimens representating liquids (serum, cell-culture supernatant, bacterial suspensions, swab samples, tap water, and rumen); faeces (pig, bird and human) as example of a complex inhibitor-rich matrix; organs like brain, heart, liver, lymph nodes, kidney, lung, and intestine to test the efficiency of the protocol on tissue; pools of midges and ticks, respectively, representing arthropod vectors; rocket, mushrooms, ham, meat loaf, pizza, strawberries as examples for different foods. In addition, TissueLyser and cryoPREP disintegrations were compared using goat lymph nodes and intestine from animals infected with Mycobacterium avium paratuberculosis (MAP; lymph nodes and intestine) that was available from an approved (Committee on the Ethics of Animal Experiments and the Protection of Animals of the State of Thuringia, Germany; Permit Number: 04-002/12) and previously published animal trial carried out in accordance with relevant guidelines and regulations^{49 (link)}.
We used samples containing a pre-diagnosed pathogen (see Table 2) and samples with unrecognized pathogen content (see Table 3). The known pathogens comprised in the samples represented the groups eukaryotic parasites, bacterial pathogens, and viruses. In addition, bacterial suspensions of exponentially growing Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, representing Gram-positive and Gram-negative bacteria, respectively, and an endospore suspension of B. subtilis as example of nucleic acids protected by highly resistant envelopes were processed. For selected samples, a sequencing library was generated according to the Supplementary File 1 (Procedure, steps 48–120) and sequenced following the respective manufacturer’s instructions.
In addition, we sequenced selected consumables used in our workflow to investigate their impact on the final sequencing outcome. The samples are an RNeasy column taken from the RNeasy Kit (Qiagen), the DNase (Qiagen) as used for the workflow, and the enzymes from the cDNA synthesis kit (Roche). The latter are the components “vial 2” (AMV RT), “vial 4” (Protector RNase Inhibitor), “vial 10” (2^nd strand enzyme) and “vial 11” (T4 DNA Polymerase). For all samples, we extracted RNA as described and DNA using the QIAamp DNA Mini Kit (Qiagen) and prepared libraries as described in the Supplementary File 1. In addition, as a blank control, an 800-µl water sample (Roth) was processed with the present workflow.

The virulence of one strain of B. bassiana (BbZJ1) to locusts treated with bacterial supplementation with three bacterial strains, separately or together as a mixture, was assessed. Before fungal and bacterial inoculation, the prothoracic sclerites of locusts were either pre-sterilized or not. For tests with bacteria collected from the outside of locust bodies, we evenly applied 10 μL of a suspension of three recovered bacterial lines (LM 5-2, LM 5-4, and LM 5-13) or each strain as a separate treatment at 10⁵ bacterial spores/mL. Then, fungal spores were applied. Locusts receiving treatments had either natural bacterial flora or were locusts whose prothoracic sclerite had been sterilized.
For tests with bacteria recovered from intestinal tissue, locusts were fed wheat leaves treated with Pectobacterum sp. or Enterobacter sp. (10⁵ spores/mL) after the locusts were first inoculated with B. bassiana on the prothoracic sclerite. Locusts in this group were fed bacteria on wheat leaves daily. Each treatment was repeated 3 times with 20 fifth-instar nymphs as one replicate. Locusts were then reared under the same conditions mentioned above, and the number of dead locusts was recorded daily.

The spores of the bacterial strain Bacillus sp. AMD05 were inoculated into 50 mL of YEME liquid medium in a 125 mL flask. The culture was incubated at 200 rpm at 30 °C for two days. After incubation, 10 mL of the AMD05 liquid culture was inoculated into a 500 mL Erlenmeyer flask containing 200 mL of YEME medium and shaken at 170 rpm and 30 °C for two days. Then, 15 mL of the medium culture was transferred into 1 L of YEME medium in a 2.8 L Fernbach flask for four days at 170 rpm and 30 °C (24 ea × 1 L, total volume 24 L). The entire culture was extracted with 36 L of ethyl acetate (EtOAc). The EtOAc layer was separated using a separation funnel (capacity 3 L), and the residual water in the EtOAc layer was removed by adding anhydrous sodium sulfate. The extract was concentrated in vacuo to yield dry material. This procedure was repeated 3 times (72 L of culture in total) to yield extracted material.

Antimicrobial activity was determined with the diffusion method in a solid nutrient media of agar. Mueller–Hinton Agar (Mueller–Hinton II Agar, BBL, Cockeysville, MD, USA) was used.
Standard cultures of nonspore bacteria (all bacteria were obtained from American Type Culture Collection (ATCC))—Staphylococcus aureus (ATCC 25923; human nasal microbiota), Staphylococcus epidermidis (ATCC 12228; human skin microbiota), Enterococcus faecalis (ATCC 29212; human colonic microbiota), Escherichia coli (ATCC 25922; human colonic microbiota), Klebsiella pneumoniae (ATCC 13883; human microbiota), Pseudomonas aeruginosa (ATCC 27853; human microbiota), and Proteus vulgaris (ATCC8427; human microbiota). Bacteria were grown for 20–24 h at 35–37 °C on Mueller–Hinton Agar. The bacterial suspension was prepared from cultures of cultivated bacteria in sterile physiological sodium chloride (0.9%) solution, standardised with a McFarland standard indicator. The bacterial suspension was considered standardised when the indicator value was 0.5 (1 mL of bacterial suspension contains 1.5 × 10⁸ cells of the micro-organism).
Standard spore bacteria cultures of Bacillus cereus (ATCC 6633; soil microbiota) were grown for 7 days at 35–37 °C on Mueller–Hinton Agar. After growing the culture of spore bacteria, it was washed off the surface of the medium with a sterile physiologic solution. The prepared suspension was heated for 30 min at 70 °C and diluted with physiological saline until the spore concentration in 1 mL was between 10 × 10⁶ and 100 × 10⁶.
The standard culture of the fungus Candida albicans (ATCC 10231; human microbiota) was grown for 20 to 24 h at 30 °C for 72 h on Sabouraud agar. The fungal suspension was prepared from cultivated fungal cultures in physiological saline and standardised with a McFarland standard indicator.
A 0.5 McFarland turbidity suspension of the standard bacteria was prepared. The bottom of the Petri dishes was divided into 9 segments. The technology of reference microorganisms to Mueller–Hinton agar was used to determine the antimicrobial activity of Glycyrrhiza glabra L. and Trifolium pratense L. extracts. The disk method was used to determine the antimicrobial activity of Myristica fragrans Houtt. essential oil.