1、Gene-targeted microfluidic cultivation validated byisolation of a gut bacterium listed in HumanMicrobiome Projects Most Wanted taxaLiang Maa, Jungwoo Kima, Roland Hatzenpichlerb, Mikhail A. Karymova, Nathaniel Hubertc, Ira M. Hananc,Eugene B. Changc, and Rustem F. Ismagilova,1Divisions ofaChemistry
2、and Chemical Engineering andbGeological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125;andcDepartment of Medicine, The University of Chicago, Chicago, IL 60637Edited by Robert Haselkorn, The University of Chicago, Chicago, IL, and approved May 19, 2014 (received for r
3、eview March 20, 2014)This paper describes a microfluidics-based workflow for geneti-cally targeted isolation and cultivation of microorganisms fromcomplex clinical samples.Data sets from high-throughput sequenc-ing suggest the existence of previously unidentified bacterial taxaand functional genes w
4、ith high biomedical importance. Obtain-ing isolates of these targets, preferably in pure cultures, is crucialfor advancing understanding of microbial genetics and physiologyand enabling physical access to microbes for further applications.However, the majority of microbes have not been cultured, due
5、 inpart to the difficulties of both identifying proper growth con-ditions and characterizing and isolating each species. We de-scribe a method that enables genetically targeted cultivation ofmicroorganisms through a combination of microfluidics and on-and off-chip assays. This method involves (i) id
6、entification of cul-tivation conditions for microbes using growth substrates availableonly in small quantities as well as the correction of sampling biasusing a “chip wash” technique; and (ii) performing on-chip geneticassays while also preserving live bacterial cells for subsequentscale-up cultivat
7、ion of desired microbes, by applying recently de-veloped technology to create arrays of individually addressablereplica microbial cultures. We validated this targeted approachby cultivating a bacterium, here referred to as isolate microfluidi-cus 1, from a human cecal biopsy. Isolate microfluidicus
8、1 is, to ourknowledge, the first successful example of targeted cultivationof a microorganism from the high-priority group of the HumanMicrobiome Projects “Most Wanted” list, and, to our knowledge,the first cultured representative of a previously unidentified genusof the Ruminococcaceae family.micro
9、scale|anaerobe|aerobe|cultivate|metagenomeThis paper describes an integrated microfluidic workflow forgenetically targeted cultivation and isolation of microorganisms.Microbes play critical functional roles in diverse environmentsranging from soil and oceans to the human gut. The emergenceof culture
10、-independent techniques has provided insights intomicrobial ecology by revealing genetic signatures of unculturedmicrobial taxa (15). It also suggests that certain microbesmay impact host phenotypes such as obesity, inflammation, andgastrointestinal integrity (6, 7). This explosion of sequencingdata
11、 has presented new challenges and opportunities for mi-crobial cultivation, which is critical for allowing direct access tomicroorganisms to test hypotheses experimentally, and is cru-cial for proper taxonomic classification, functional annotationof metagenomic sequences, and use of such microbes fo
12、r en-vironmental remediation, energy applications, and formulationof probiotics. However, a direct approach that cultivates, ina targeted fashion, microbes carrying genes of interest identi-fied in metagenomic data sets remains mostly unexplored.As a result, for example, a list of the “Most Wanted”
13、taxa thatare urgently in need of cultivation has been issued by the Hu-man Microbiome Project (HMP) from the National Institutesof Health. These microorganisms are highly prevalent and abundantin the human microbiome but poorly represented in culturedcollections (2).Most microbes do not grow using t
14、raditional cultivationmethods and hence are referred to as “unculturable” (810).Although these microbes could be grown in their natural habitats(9), where effects such as cross-feeding (11) and microbehostinteractions (12, 13) are present, some biological samples, suchas clinical biopsies, are often
15、 limited in quantity. This makes itchallenging to set up cultivation experiments in large scale withthese native media, but creates opportunities for miniaturizedmethods. Further, miniaturized methods that use compartmen-talization can eliminate competition among species. Cultivationmethods that use
16、 miniaturization and compartmentalization, in-cluding gel microdroplets (14), miniaturized Petri dishes (15), andmicrofluidics (1619), have become increasingly promising as abasis for targeted microbial cultivation and isolation platforms, asthey can limit the consumption of precious samples and als
17、ocontrol the microenvironment around cells (20). We envisionedimplementing targeted cultivation with microfluidics by focusingon two goals. The first goal is to efficiently identify cultivationSignificanceObtaining cultures of microbes is essential for developingknowledge of bacterial genetics and p
18、hysiology, but manymicrobes with potential biomedical significance identified frommetagenomic studies have not yet been cultured due to thedifficulty of identifying growth conditions, isolation, and char-acterization. We developed a microfluidics-based, geneticallytargeted approach to address these
19、challenges. This approachcorrects sampling bias from differential bacterial growth kinet-ics, enables the use of growth stimulants available only in smallquantities, and allows targeted isolation and cultivation of apreviously uncultured microbe from the human cecum thatbelongs to the high-priority
20、group of the Human MicrobiomeProjects “Most Wanted” list. This workflow could be leveragedto isolate novel microbes and focus cultivation efforts on bio-medically important targets.Author contributions: L.M., J.K., R.H., E.B.C., and R.F.I. designed research; L.M., J.K., R.H.,M.A.K., N.H., and I.M.H.
21、 performed research; L.M., J.K., and R.H. analyzed data; and L.M.,R.H., and R.F.I. wrote the paper.Conflict of interest statement: R.F.I. has a financial interest in SlipChip Corporation.This article is a PNAS Direct Submission.Freely available online through the PNAS open access option.Data deposit
22、ion: The genome sequences reported in this paper have been deposited inthe Joint Genome Institutes Integrated Microbial Genomes database, https:/img.jgi.doe.gov/cgi-bin/w/main.cgi (accession no. 2545555870). The 16S rRNA gene sequences ofisolate microfluidicus 1 reported in this paper have been depo
23、sited in the GenBankdatabase (accession nos. KJ875866 and KJ875867).1To whom correspondence should be addressed. E-mail: rustem.admincaltech.edu.This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1404753111/-/DCSupplemental.97689773 | PNAS | July 8, 201
24、4 | vol. 111 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1404753111conditions that support growth of target microbes. This can beaccomplished by performing a genetic assay with target-specificprimers or probes on the pooled microbial culture from a certaincultivation condition before isolation (21);
25、however, designingspecific probes based on short reads from high-throughput se-quencing can be difficult. Moreover, it can be challenging todetect and cultivate slowly growing strains, as they often fallbelow the limit of detection, being outcompeted by rapidlygrowing strains in a complex community.
26、 A second goal of tar-geted cultivation is to focus isolation efforts on microbial targetsof interest, thereby minimizing the effort associated with iso-lating off-target colonies. However, both PCR and fluorescencein situ hybridization (FISH) require access to genetic material,which is often not co
27、mpatible with the goal of isolating andcultivating live cells. This paper addresses these challenges. Inan accompanying paper (22), we describe the design, fabrication,and underlying physics of a microfluidic device to create arraysof individually addressable replica microbial cultures. Here, weinte
28、grate this device and additional devices and methods intoa workflow for genetically targeted microbial cultivation, andvalidate this workflow by isolating a bacterium from the MostWanted taxa.Results and DiscussionOverview of Workflow for Genetically Targeted Microfluidics-BasedCultivation. We envis
29、ioned isolating and cultivating microbialtargets identified from metagenomic or 16S ribosomal RNA (16SrRNA) gene high-throughput sequencing studies by combiningmicrofluidics with genetic assays (Fig. 1A). To address thegoal of streamlining cultivation efforts using genetic assays, wecreated a genera
30、l workflow with two major components: identi-fication of cultivation conditions for the target organism (Fig. 1B)and isolation of the target (Fig. 1C). In both components, singlebacterial cells from clinical samples are stochastically con-fined in nanoliter wells on a microfluidic device to promote
31、thegrowth of microcolonies. This confinement can be useful forsuppression of overgrowth from rapidly growing strains, in favorof slowly growing strains. In the first step, a “chip wash” methodis used to monitor bacterial growth on a microfluidic device (Fig.1B) under various conditions; miniaturizat
32、ion allows cultivationexperiments that involve limited quantities of natural growthstimulants. In this method, microcolonies grown under eachcultivation condition are collected into a single tube by washingthe microwells after cultivation, analogously to the plate washPCR method (21). DNA from the p
33、ooled cells is analyzed bysequencing, target-specific primers, or both, to determine whetherthe cultivation conditions for that chip allowed the growth ofthe target microorganism. This chip wash method can be re-peated sequentially or in parallel until the growth conditionsare identified. Then, the
34、target organism is isolated and culti-vated (Fig. 1C): The sample is cultivated on a separate micro-fluidic device, described in an accompanying paper (22), underthe optimal condition identified during chip wash. After cul-tivation, this device splits each microcolony into two identicalcopies. We an
35、ticipate that multiple rounds of culture and split-ting on the same device could be performed in a similar fashion.PCR is performed on the first copy to identify the compartmentcontaining the target of interest, and then live cells can be retrievedfrom the corresponding well on the other half of the
36、 chip forscale-up cultivation.To implement this workflow, we relied on the SlipChip plat-form for three reasons (23). First, it can create thousands ofminiaturized reactions without the need for bulky equipment. Itcan be used in the limited space of an anaerobic chamber, whichis widely used to culti
37、vate anaerobes that dominate the humangut microbiota. Second, SlipChip is compatible with PCR (24)and enzymatic assays (25). Third, compartmentalization onSlipChip is reversible and the microcolonies can be spatiallyindexed as described in an accompanying paper (22), whichfacilitates the retrieval o
38、f reagents and organisms from thedevice (24, 26).Chip Wash Device. Fig. 2A shows the general workflow of a chipwash experiment. We designed a microfluidic device to performup to 3,200 microbial cultivation experiments, each on a scale of6 nL (Fig. 2C and SI Appendix). This device enables threecapabi
39、lities: stochastic confinement of single cells from samples,microbial cultivation, and collection of cultivated cells. To con-fine single cells, a sample of bacteria suspended in cultivationmedium is loaded into the channels and wells (Fig. 2A, ii).Slipping the bottom plate (dashed layer in Fig. 2A)
40、 upwardenables stochastic confinement of bacterial cells in wells (Fig. 2A, iii). To introduce gas into the channel and remove residualsample in the channel, the solution is purged from the channel byvacuum (Fig. 2A, iv). To cultivate microbes, the device is in-cubated and some of the single cells g
41、row to microcolonies (Fig.2A, v). After cultivation, the microchannel is loaded with buffersolution (Fig. 2A, v) to avoid the formation of gas bubbles. Thepresence of gas bubbles in a channel could increase flow re-sistance (27) and therefore slow down or stop the flow in thatchannel, resulting in i
42、nefficient washing in later steps. To allowcollection of the microbial cells, the bottom plate is slipped backto overlay the wells with the channel (Fig. 2A, vi). A buffer so-lution is injected to flush the channel (Fig. 2A, vii) and is col-lected, from the outlet specifically designed for collectio
43、n (Fig. 2A, viii and C), in a pipette tip. The flow of fluid on SlipChip isPooled bacterial cells.ATTGCA.CTGGCA.GTGGTA.GTGGTA.Chip Washand collectB Chip washSequencingABCDA1B1C1D1A2B2C2D2C Splitting and PCRConfirming ConfirmingtargetMetagenomic data53535353BacterialsuspensionStochasticconfinementtar
44、getABCDA SequencingPCRA2B2C2D2Scale up PCRs98764532SplitGrow colonies Grow coloniesFig. 1. Illustration representing the workflow for targeted cultivation andisolation of microbial organisms. (A) Microbial targets carrying genes of in-terest are identified by high-throughput sequencing of clinical s
45、amples. Arepresentative sequence of the target is shown in red. To cultivate the tar-get, the inoculum is suspended in cultivation medium and loaded ontoa microfluidic device, enabling stochastic confinement of single cells andcultivation of individual species (represented by different shapes). (B)
46、A chipwash method is used to monitor bacterial growth under different cultivationconditions. Cells are pooled en masse into a tube and DNA is extracted forgenetic analysis such as sequencing and PCR. (C) The target can be isolatedby growing the sample under the growth condition identified from the c
47、hipwash. The two halves of the device are separated, resulting in two copies ofeach colony. On one half of the chip, target colonies are identified usingPCR. Then, the target colony on the other half of the chip is retrieved fora scale-up culture, after which sequencing is used to validate that the
48、correcttarget has been isolated.Ma et al. PNAS | July 8, 2014 | vol. 111 | no. 27 | 9769ENGINEERINGcontrolled by positive pressure using a pipettor. This process ofinjectioncollection is repeated three times. Immiscible oil isthen injected to further displace the remaining aqueous phase.We used a re
49、d dye experiment to visualize the device operationdescribed above (Fig. 2B), which allowed us to observe thatthe droplets remained intact during purging when gas was in-troduced into the channels. In addition, in the chip wash step,the solutions from the channel and the wells were merged andcould be visualized by the originally colorless solutions fromthe channel turning red. The removal of red dye can be ob-served in Fig. 2B, vii as the solution in the channel turned backto colorless. To quantify the recovery efficiency of this method,a solution with a fluorescent dye
