In situ cultivation of previously uncultivable microorganisms using the ichip

Abstract

Most microbial species remain uncultivated, and modifying artificial nutrient media brings only an incremental increase in cultivability. We reasoned that an alternative way to cultivate species with unknown requirements is to use naturally occurring combinations of growth factors. To achieve this, we moved cultivation into the microbes' natural habitat by placing cells taken from varying environmental samples into diffusion chambers, which are then returned to nature for incubation. By miniaturizing the chambers and placing only one to several cells into each chamber, we can grow and isolate microorganisms in axenic culture in one step. We call this cultivation platform the 'isolation chip', or 'ichip'. This platform has been shown to increase microbial recovery from 5- to 300-fold, depending on the study. Furthermore, it provides access to a unique set of microbes that are inaccessible by standard cultivation. Here we provide a simple protocol for building and applying ichips for environmental cultivation of soil bacteria as an example; the protocol consists of (i) preparing the ichip; (ii) collecting an environmental sample; (iii) serially diluting cells and loading them into the ichip; (iv) returning the ichip to the environment for incubation; (v) retrieving the ichip and harvesting grown material; and (vi) domestication of the ichip-derived colonies for growth in the laboratory. The ichip's full assembly and deployment is a relatively simple procedure that, with experience, takes ∼2-3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take ∼1-4 h, depending on which specific procedures are used.

Cultivatingin situtriggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

“…Various in situ cultivation methods with similar basic rationales have been applied to various environmental samples and have been shown to be highly capable of microbial cultivation compared with conventional approaches because the method led to isolating some uncultivated and phylogenetically new and industrially important isolates (18-21, 51–53). Furthermore, mining “microbial dark matter” using this new approach remains highly expected (2).…”

Thinking Outside the Box—Novel Antibacterials To Tackle the Resistance Crisis

“…[24,27,28] Ar ecent successful example is the coculturing of two marine species,t he producer Micromonospora together with Rhodococcus sp., to afford the bis-nitroglycosylated anthracycline keyicin ( Figure 3). [35] Thefirst antibiotic discovered using aminiaturized, multiwell diffusion chamber called iChip (isolation chip) was lassomycin ( Figure 3), an inhibitor of the ATP-dependent protease complex ClpC1ClpP1P2 in M. tuberculosis. [29] To dive deep into the pool of uncharted, yet promising natural products,s cientists have now shifted their focus towards exploring the "microbial dark matter", [30] namely,the 85-99 %o fb acteria and archaea that defy cultivation in the laboratory.…”

“…[33,34] Importantly,o nce ac olony is produced in situ, as ubstantial number of isolates can also be cultivated under laboratory conditions,t hereby allowing the investigation of antibacterial natural products. [35] Thefirst antibiotic discovered using aminiaturized, multiwell diffusion chamber called iChip (isolation chip) was lassomycin ( Figure 3), an inhibitor of the ATP-dependent protease complex ClpC1ClpP1P2 in M. tuberculosis. [36] Despite the advances described, the cultivation of many microbes is still amajor obstacle.Inthis regard, metagenomic approaches offer ameans for identifying new antibiotics and their respective biosynthetic gene clusters (BGCs) without the need to cultivate the original producer strain (Figure 3c).…”

Über bisherige Denkweisen hinaus – neue Wirkstoffe zur Überwindung der Antibiotika‐Krise

“…In Anerkennung der wegweisenden Arbeiten des Nobelpreisträgers Selman Waksman bezeichnet man diese Strategie auch als die Waksman-Plattform. [35] Das erste Antibiotikum, das mithilfe einer miniaturisierten, Multiwell-Diffusionskammer,d es iChips (Isolations-Chips) entdeckt wurde,w ar Lassomycin (Abbildung 3), ein Inhibitor des ATP-abhängigen Proteasekomplexes ClpC1ClpP1P2 in M. tuberculosis. Dabei stellt vor allem die Wiederentdeckung bereits bekannter Naturstoffe ein entscheidendes Hindernis dar, welches zu einem drastischen Anstieg der Ausfallrate bei der Naturstoffentdeckung geführt hat.…”

“…[33,34] Entscheidend dabei ist, dass eine Vielzahl natürlicher Isolate auch unter Laborbedingungen kultiviert und auf die Produktion antibakterieller Naturstoffe untersucht werden kann, sobald sich eine Kolonie erst einmal in situ etabliert hat. [35] Das erste Antibiotikum, das mithilfe einer miniaturisierten, Multiwell-Diffusionskammer,d es iChips (Isolations-Chips) entdeckt wurde,w ar Lassomycin (Abbildung 3), ein Inhibitor des ATP-abhängigen Proteasekomplexes ClpC1ClpP1P2 in M. tuberculosis. [36] Tr otz der beschriebenen Fortschritte stellt die Kultivierung zahlreicher Mikroorganismen immer noch eine wesentliche Hürde dar.…”