This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.
A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.
Assessing fitness and virulence of pathogenic organisms is a fundamental aspect of microbiology research. It enables comparisons to be made between strains or between mutated organisms, which allows researchers to determine the importance of certain genes under specific conditions. Traditionally, virulence assessment utilizes an animal model of infection using different bacterial strains and observing the outcome of the infected animal (e.g. Infectious Dose50, Lethal Dose50, time to death, symptom severity, lack of symptoms, etc.). This procedure provides valuable descriptions of virulence, but it requires strains to cause considerable differences in outcomes in order to detect variations from wild-type. Furthermore, results are semi-quantitative because while disease progression and symptom severity can be subjectively quantified over time, interpretation of virulence compared to wild-type is more qualitative (i.e. more, less, or equally virulent). A common alternative to performing animal infectivity assays is to generate competitive indices (CIs), values that directly compare fitness or virulence of a strain to a wild-type counterpart in a mixed infection1. This technique has numerous advantages over a traditional animal model of infection by standardizing virulence to a wild-type strain and determining a quantifiable value to reflect the degree of attenuation. This technique can also be adapted to analyze gene interactions in bacteria by determining a canceled out competitive index (COI)2. Calculating a COI for a group of mutated organisms allows researchers to determine whether two genes independently contribute to pathogenesis or if they are involved in the same virulence pathway and dependent on each other. Additionally, calculating a CI requires enumeration of bacteria which can provide valuable insights into the pathogenesis of organisms. CIs and COIs also allow researchers to asses avirulent strains that do not cause clinical disease but still have differences in fitness. This technique is limited by the use of traditional antibiotic resistance markers to identify strains, thereby limiting the number of input strains to only one or two at a time. Because of this limitation, large numbers of experimental groups and replicates are required, which in addition to adding to labor and material costs, also increases opportunities for variability in experimental conditions and inaccurate results. (For a thorough review of the benefits and applications of using mixed infections to study virulence, fitness, and gene interactions, see C.R. Beuzón and D.W. Holden 1)
Attempts have been made to overcome this limitation, such as the use of fluorescently-labeled cells quantified via flow cytometry3,4,5. This technique quantifies cells using either 1) labeled antibodies to phenotypic markers or 2) endogenously produced fluorescent proteins. The use of labeled antibodies has a limit of detection of 1,000 cells/mL, and therefore requires a high number of cells to analyze3. Cells expressing fluorescent proteins have an altered physiology and are susceptible to fitness changes resulting from high protein expression6. Both methods are limited by the number of fluorescent markers detectable using flow cytometry. An advancement in molecular quantification was achieved through the development of a microarray technique that detected attenuation in 120 strains from an initial mixed infection of over 1,000 strains in a murine model7. This technique utilized a microarray analysis of RNA from mutated strains, which lead to considerable variability in the outcome. Nevertheless, it established that large pools of mixed infections can be a useful tool and that by utilizing sensitive detection techniques, differences in bacterial virulence can be identified. With the development of the next generation sequencing, Tn-seq expanded the utility of transposon mutations, enabling a powerful method for quantifying bacteria that were randomly mutated8,9,10,11. An alternative protocol was recently developed that eliminates the need for transposons and instead uses DNA barcodes to more easily identify and track genomic changes and their impact on fitness12. This technology is a major advancement, but the insertion of the genomic barcodes is still a random process. To overcome the randomness of previous experiments, Yoon et al. developed a method to calculate the CIs of Salmonella strains using unique DNA barcodes inserted at precise locations on the chromosomes of bacteria13. Unique barcoded strains were detected using a qPCR-based method with SYBR green and primers specific to each unique barcode. The technique was limited by constraints imposed by qPCR, including differences in primer efficiencies and low sensitivity, evidenced by the need for nested-PCR prior to qPCR. Nevertheless, this approach demonstrated that targeted genomic modifications could be exploited for detecting and potentially quantifying pools of multiple bacterial strains.
In the following protocol, we describe a novel methodology to perform bacterial competition experiments with large pools of mixed inocula followed by accurate quantification using a highly sensitive digital PCR technique. The protocol involves genetically-labeling bacterial strains with a unique DNA barcode inserted on an innocuous region of the chromosome. This modification allows strains to be quickly and accurately quantified using modern molecular technology instead of traditional serial dilutions, replica plating, and counting colony forming units that rely on phenotypic markers (i.e. antibiotic resistance). The modifications allow for simultaneous assessment of many strains in a single pooled inoculum, substantially reducing the possibility of experimental variability because all strains are exposed to the exact same conditions. Furthermore, while this technique was developed in Salmonella enterica serovar Typhimurium, it is highly adaptable to any genetically malleable organism and nearly any experimental design where accurate bacterial counts are required, providing a new tool to increase accuracy and throughput in microbiology laboratories without the constraints imposed by previous methods.
The ability to accurately quantify microorganisms is of paramount importance to microbiology research, and the ability to enumerate unique strains from an initial mixed population has proved to be an invaluable tool for assessing fitness and virulence traits in bacteria. However, the techniques for accomplishing this have not progressed in pace with modern developments in molecular biology. The technology to easily modify the chromosomes of many bacteria, including S. Typhimurium, has been available for nearly t…
The authors have nothing to disclose.
Research reported in this publication was supported by the George F. Haddix President’s Faculty Research Fund and the National Institute of General Medical Science of the National Institutes of Health (NIH) under award number GM103427. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
1.5 mL microcentrifuge tubes | Eppendorf | 22600028 | Procure from any manufacturer |
16 mL culture tubes | MidSci | 8599 | Procure from any manufacturer |
5-200 μL pipette tips | RAININ | 30389241 | Procure alternative tip brands with caution based on manufacturing quality |
5-50 μL multichannel pipette | RAININ | 17013804 | Use alternative multichannel pipettes with caution |
Agarose | ThermoFisher Scientific | BP160-500 | Procure from any manufacturer |
BLAST Analysis | NCBI | N/A | https://blast.ncbi.nlm.nih.gov/Blast.cgi |
C1000 Touch Thermocycler with 96-Deep Well Reaction Module | Bio Rad | 1851197 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
Chemically competent DH5α | Invitrogen | 18258012 | Procure from any manufacturer or prepare yourself |
Chloramphenicol | ThermoFisher Scientific | BP904-100 | Procure from any manufacturer |
Cytation5 Microplate reader | BioTek | CYT5MF | Procure from any manufacturer, use any system capable of accurately quantifying DNA |
Data Analysis Software (QuantaSoft and QuantaSoft Data Analysis Pro) | Bio Rad | N/A | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
ddPCR 96-Well Plates | Bio Rad | 12001925 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
ddPCR Droplet Reader Oil | Bio Rad | 1863004 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
ddPCR Supermix for Probes (No dUTP) | Bio Rad | 1863024 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
DG8 Cartridges for QX200/QX100 Droplet Generator | Bio Rad | 1864008 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
DG8 Gaskets for QX200/QX100 Droplet Generator | Bio Rad | 1863009 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
Droplet Generation Oil for Probes | Bio Rad | 1863005 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
Kanamycin | ThermoFisher Scientific | BP906-5 | Procure from any manufacturer |
Luria-Bertani agar | ThermoFisher Scientific | BP1425-2 | Procure from any manufacturer or make it yourself from agar, tryptone, yeast digest, and NaCl |
Luria-Bertani broth | ThermoFisher Scientific | BP1426-2 | Procure from any manufacturer or make it yourself from tryptone, yeast digest, and NaCl |
PCR Plate Heat Seal, foil, pierceable | Bio Rad | 1814040 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
PCR Tubes | Eppendorf | 951010022 | Procure from any manufacturer |
Petri dishes | ThermoFisher Scientific | FB0875712 | Procure from any manufacturer |
pPCR Script Cam SK+ | Stratagene/Agilent | 211192 | No longer available commercially |
Primer/Probe Design | IDT | N/A | https://www.idtdna.com/Primerquest/Home/Index |
pSKAP and pSKAP_Barcodes | Addgene | Plasmid numbers 122702-122726 | www.addgene.org |
PX1 PCR Plate Sealer | Bio Rad | 1814000 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
QX200 Droplet Generator | Bio Rad | 1864002 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
QX200 Droplet Reader | Bio Rad | 1864003 | Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available. |
S. Typhimurium strain ATCC 14028s | ATCC | ATCC 14028s | www.atcc.org |
Take3 Micro-Volume Plate | BioTek | TAKE3 | Procure from any manufacturer, use any system capable of accurately quantifying DNA |
Thermo Scientific FastDigest BamHI | ThermoFisher Scientific | FERFD0054 | Procure from any manufacturer |
Thermo Scientific FastDigest DpnI | ThermoFisher Scientific | FERFD1704 | Procure from any manufacturer |
Thermo Scientific FastDigest HindIII | ThermoFisher Scientific | FERFD0504 | Procure from any manufacturer |
Thermo Scientific GeneJet Gel Extraction and DNA Cleanup Micro Kit | ThermoFisher Scientific | FERK0832 | Procure from any manufacturer |
Thermo Scientific GeneJet Miniprep Kit | ThermoFisher Scientific | FERK0503 | Procure from any manufacturer |
Thermo Scientific Phusion High-Fidelity DNA Polymerase | ThermoFisher Scientific | F534L | Procure from any manufacturer |
Thermo Scientific T4 DNA Ligase | ThermoFisher Scientific | FEREL0011 | Procure from any manufacturer |
Thermocycler | Bio Rad | 1861096 | Procure from any manufacturer |
UVP Visi-Blue Transilluminator | ThermoFisher Scientific | UV95043301 | Or other transiluminator that allows visualization of DNA |
Water, Molecular Biology Grade | ThermoFisher Scientific | BP28191 | Procure from any manufacturer |