Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Summary

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.

Abstract

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.

Introduction

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 cytometry^3,4,5. 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 mutated^8,9,10,11. 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.

Protocol

1. Incorporate Unique DNA Barcodes onto a Plasmid Containing the Necessary Components for Allelic Exchange NOTE: A new plasmid, named pSKAP, with a high copy number and increased transformation efficiency compared to the existing pKD13 allelic exchange plasmid was created. This is described in steps 1.1-1.12 (Figure 1). The finalized plasmids containing unique DNA barcodes and components for allelic exchange are available through a plasmid repositor…

Representative Results

The use of this methodology requires that appropriate control reactions are performed to validate the sensitivity and specificity of each probe used to identify target DNA. In this representative experiment, we validated eight unique DNA barcodes with the eight corresponding probes for identification. All eight probes had a low rate of false positives in both NTC and negative control reactions (Table 3), highlighting their specificity even among highly similar DNA sequenc…

Discussion

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…

Materials

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