Research reported in this publication was supported by the George F. Haddix President&#8217;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.

<ol>
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The authors have nothing to disclose.

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 two decades14, yet this ability has been rarely utilized for molecularly tagging strains with unique DNA sequences. By exploiting the ability to create readily identifiable strains based on unique, minimally-disruptive DNA barcodes inserted onto the bacterial chromosome, coupled with the most state-of-the-art technology to detect and quantify these molecular identifiers (i.e. digital PCR), we have created a system that provides exquisite sensitivity, specificity, accuracy, and precision for easily quantifying individual strains within a diverse population of bacteria.
Calculating CIs and COIs as described above relies on the ability to accurately make the appropriate modifications to bacterial chromosomes. All modifications should be verified by Sanger sequencing to ensure that no random mutations occurred. The use of a high-fidelity polymerase will minimize these errors, but any such mutations that do occur will impair the ability to detect the strain, which is another critical aspect of this protocol. Although we have demonstrated that digital PCR can detect as few as two gDNA copies in a background of nearly 2 million, strains outside of this range may require additional dilutions for their accurate quantification. Furthermore, DNA barcode sequences must be designed to facilitate the use of high-quality probe sequences. Probe sequences should be analyzed for tendencies to self-dimerize, form hairpins, or ineffectively bind their targets. The importance of using quality sequences and probes cannot be minimalized, a fact that is evidenced by the careful validation experiments that must be performed with each probe. Efforts to create optimal DNA barcodes will create optimal digital PCR quantification results.
While carefully designed molecular tags are important for obtaining quality results, interpreting the results is another critical aspect of this protocol. The CI is defined as the ratio between the mutant strain and the wild-type strain in the output divided by the ratio of the two strains in the input1,19,20. This traditional CI presented in section 9 is useful when the mixed infection consists of only one strain versus wild-type. However, when using large pools of strains to inoculate media or animals, strains are not only competing against wild-type, but also against every other strain present in the inoculum. Previous studies that performed competition experiments using multiple infecting strains have failed to take this into account in their calculations7,13. To account for this feature of mixed infections, we have introduced a formula to calculate CIs that has been modified for pooled infections. It is unlikely that all strains will provide the same level of competition a wild-type strain would. However, because most bacterial genes have little impact on virulence, as the number of strains used in competitive index experiment increases, the likelihood that overall virulence will tend toward the wild-type strain increases. This may not necessarily be the case for certain experimental designs using pools of many strains all with known virulence defects. However, this is accounted for in the modified equation because less abundant strains (less fit) in the outcome will have a smaller effect on Xoutput. Depending on the specific experimental design, there may be cases in which one or the other formula is preferred. In most instances involving pooled infections, however, it is important to consider that in a mixed infection, all strains compete against each other, not just against wild-type. When analyzing results, it is critical that the rationale behind each formula is well-understood to make the most accurate interpretations of strain fitness. When reporting results, it is equally important to accurately disclose how data were analyzed.
Section 9 of the protocol also includes formulas to help determine gene interactions using a COI. With this analysis, predictions can be made to determine whether two virulence genes operate independently or together. COI is defined as the ratio of the double mutant to single mutant strain in the output divided by the ratio of the two strains in the input1. The formula is designed to detect phenotypic additivity of gene disruptions. If genes function independently to enhance virulence, a disruption of both genes should cause a greater decrease in fitness compared to a single disruption of either gene alone. If genes function together to enhance virulence (such as genes encoding two enzymes in a pathway), a disruption of either gene should have the same effect on virulence as disrupting both genes. Detecting phenotypic additivity can be difficult in cases where the level of attenuation caused by a single gene is either very high or very low. Nevertheless, direct comparison of strains within the same animal system provides less variability and a more reliable account of the functional relationship between genes, and this calculation can be performed from two strains within a larger mixed population.
A final critical aspect for interpreting results is to consider the effects of population dynamics. In some mixed infections that have multiple strains, a single strain may emerge as either more dominant or less fit because of random population drift. This phenomenon can be amplified when bottleneck events occur. This can be caused from using a very large number of input strains, a very small number of total bacteria in the inoculum, or a combination of both. Another interfering aspect that arises from mixed infections is the possibility of in trans complementation. This occurs when a fit strain, such as the wild-type, artificially enhances the virulence of a less fit strain. A hypothetical example of this would be to compare the fitness of an S. Typhimurium Pathogenicity Island 2 (SPI2)-knockout strain co-infected with a wild-type strain. SPI2 enables S. Typhimurium to survive intracellularly by secreting effectors into the host cytosol that modify the phagosome within a macrophage. Disruption of this system makes S. Typhimurium susceptible to intracellular killing. However, because macrophages are capable of engulfing two or more bacteria at once, the SPI2-knockout could receive a considerable increase in fitness if it is residing in the same macrophage as a wild-type S.&#160;Typhimurium that is secreting effectors into the host cytosol. Random population dynamics and the possibility of in trans complementation is a limitation of any competition experiment. If in trans complementation is suspected, phenotypes should be confirmed using other complementary methods to assess fitness. To overcome random population dynamics, increasing the number of replicate experiments increases the likelihood of identifying outliers in results. Fortunately, the protocol described above makes it easier to have a greater number of identical replicate experiments because the number of experimental conditions is drastically reduced.
A key element to the CI technique described above is its ability to be adapted to almost any organism and any experimental design that requires accurate quantification of microorganisms. It does, however, require the genetic manipulation of an organism to incorporate a unique DNA sequence on the chromosome. The adaptability of the technique requires the species to be genetically-malleable and will rely on the generation of alternative protocols for modifying the genome (Steps 1 and 2). The DNA barcodes listed in Tables 2 and S1 should be enough for most bacteria; however, as in all quantitative PCR experiments, it is pertinent to analyze the bacterial genome to ensure minimal potential for off-target binding of primers and probes by a simple BLAST analysis. The DNA barcode sequences used in this study differ by only 3-4 bases in some cases, highlighting the exquisite specificity of the probes that minimizes the potential for non-specific binding. Regardless of fluorescent probes&#8217; specificity, all new barcode sequences must be appropriately validated for sensitivity and specificity as described in step 6. After creating barcoded strains, it is possible to adapt this protocol to many types of experiments besides in vitro competition assays as described above. The strains are suitable for in vivo competition assays in mice or other animal model systems. Only minimal modifications are required to extract gDNA from animal organs and tissues, and these modifications are well described by manufacturers of kits for such purposes. Further, large pools of mixed infections for in vivo competition have been successfully utilized previously7, offering the potential to reduce the number of animals necessary for a single experiment, which not only decreases costs of those experiments but also decreases the potential for animal-to-animal variability. Similarly, barcoded strains could be used in other in vitro assays that examine the susceptibility of strains to various treatment conditions (e.g. antibiotics, acid susceptibility, killing by reactive oxygen or nitrogen species, etc.). For these experiments, assessing growth inhibition could be achieved by adding the desired chemical to the growth medium in step 3.4 and proceeding as described. To assess bacterial death, a large pool of strains could be mixed, and the input quantified as described above. After exposure to the desired treatment, live cells could be selectively quantified by coupling the digital PCR quantification procedure with Viability PCR to differentiate between live and dead cells21,22,23,24,25,26. Throughput for such experiments would be dramatically increased because dilutions and replica plates for each strain are replaced by more streamlined molecular techniques. Lastly, although analyzing evolutionary biology and population genetics is beyond the scope of this paper, barcoded organisms are highly adaptable for such studies.&#160; Ultimately, the purpose of this protocol was to develop a powerful technique for quantifying bacteria that is highly adaptable for its use in many diverse species and in many types of experiments.

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. Beuz&#243;n 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.&#160; 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.

<table><tbody><tr><td>1.5 mL microcentrifuge tubes</td><td>Eppendorf</td><td>22600028</td><td>Procure from any manufacturer</td></tr><tr><td>16 mL culture tubes</td><td>MidSci</td><td>8599</td><td>Procure from any manufacturer</td></tr><tr><td>5-200 &amp;mu;L pipette tips</td><td>RAININ</td><td>30389241</td><td>Procure alternative tip brands with caution based on manufacturing quality</td></tr><tr><td>5-50 &amp;mu;L multichannel pipette</td><td>RAININ</td><td>17013804</td><td>Use alternative multichannel pipettes with caution</td></tr><tr><td>Agarose</td><td>ThermoFisher Scientific</td><td>BP160-500</td><td>Procure from any manufacturer</td></tr><tr><td>BLAST Analysis</td><td>NCBI</td><td>N/A</td><td>https://blast.ncbi.nlm.nih.gov/Blast.cgi</td></tr><tr><td>C1000 Touch Thermocycler with 96-Deep Well Reaction Module</td><td>Bio Rad</td><td>1851197</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>Chemically competent DH5&amp;alpha;</td><td>Invitrogen</td><td>18258012</td><td>Procure from any manufacturer or prepare yourself</td></tr><tr><td>Chloramphenicol</td><td>ThermoFisher Scientific</td><td>BP904-100</td><td>Procure from any manufacturer</td></tr><tr><td>Cytation5 Microplate reader</td><td>BioTek</td><td>CYT5MF</td><td>Procure from any manufacturer, use any system capable of accurately quantifying DNA</td></tr><tr><td>Data Analysis Software (QuantaSoft and QuantaSoft Data Analysis Pro)</td><td>Bio Rad</td><td>N/A</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>ddPCR 96-Well Plates</td><td>Bio Rad</td><td>12001925</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>ddPCR Droplet Reader Oil</td><td>Bio Rad</td><td>1863004</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>ddPCR Supermix for Probes (No dUTP)</td><td>Bio Rad</td><td>1863024</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>DG8 Cartridges for QX200/QX100 Droplet Generator</td><td>Bio Rad</td><td>1864008</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>DG8 Gaskets for QX200/QX100 Droplet Generator</td><td>Bio Rad</td><td>1863009</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>Droplet Generation Oil for Probes</td><td>Bio Rad</td><td>1863005</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>Kanamycin</td><td>ThermoFisher Scientific</td><td>BP906-5</td><td>Procure from any manufacturer</td></tr><tr><td>Luria-Bertani agar</td><td>ThermoFisher Scientific</td><td>BP1425-2</td><td>Procure from any manufacturer or make it yourself from agar, tryptone, yeast digest, and NaCl</td></tr><tr><td>Luria-Bertani broth</td><td>ThermoFisher Scientific</td><td>BP1426-2</td><td>Procure from any manufacturer or make it yourself from tryptone, yeast digest, and NaCl</td></tr><tr><td>PCR Plate Heat Seal, foil, pierceable</td><td>Bio Rad</td><td>1814040</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>PCR Tubes</td><td>Eppendorf</td><td>951010022</td><td>Procure from any manufacturer</td></tr><tr><td>Petri dishes</td><td>ThermoFisher Scientific</td><td>FB0875712</td><td>Procure from any manufacturer</td></tr><tr><td>pPCR Script Cam SK&lt;sup&gt;+&lt;/sup&gt;</td><td>Stratagene/Agilent</td><td>211192</td><td>No longer available commercially</td></tr><tr><td>Primer/Probe Design</td><td>IDT</td><td>N/A</td><td>https://www.idtdna.com/Primerquest/Home/Index</td></tr><tr><td>pSKAP and pSKAP_Barcodes</td><td>Addgene</td><td>Plasmid numbers 122702-122726</td><td>www.addgene.org</td></tr><tr><td>PX1 PCR Plate Sealer</td><td>Bio Rad</td><td>1814000</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>QX200 Droplet Generator</td><td>Bio Rad</td><td>1864002</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>QX200 Droplet Reader</td><td>Bio Rad</td><td>1864003</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>&lt;em&gt;Salmonella typhimurium&lt;/em&gt; strain ATCC 14028s</td><td>ATCC</td><td>ATCC 14028s</td><td>www.atcc.org</td></tr><tr><td>Take3 Micro-Volume Plate</td><td>BioTek</td><td>TAKE3</td><td>Procure from any manufacturer, use any system capable of accurately quantifying DNA</td></tr><tr><td>Thermo Scientific FastDigest BamHI</td><td>ThermoFisher Scientific</td><td>FERFD0054</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific FastDigest DpnI</td><td>ThermoFisher Scientific</td><td>FERFD1704</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific FastDigest HindIII</td><td>ThermoFisher Scientific</td><td>FERFD0504</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific GeneJet Gel Extraction and DNA Cleanup Micro Kit</td><td>ThermoFisher Scientific</td><td>FERK0832</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific GeneJet Miniprep Kit</td><td>ThermoFisher Scientific</td><td>FERK0503</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific Phusion High-Fidelity DNA Polymerase</td><td>ThermoFisher Scientific</td><td>F534L</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific T4 DNA Ligase</td><td>ThermoFisher Scientific</td><td>FEREL0011</td><td>Procure from any manufacturer</td></tr><tr><td>Thermocycler</td><td>Bio Rad</td><td>1861096</td><td>Procure from any manufacturer</td></tr><tr><td>UVP Visi-Blue Transilluminator</td><td>ThermoFisher Scientific</td><td>UV95043301</td><td>Or other transiluminator that allows visualization of DNA</td></tr><tr><td>Water, Molecular Biology Grade</td><td>ThermoFisher Scientific</td><td>BP28191</td><td>Procure from any manufacturer</td></tr></tbody></table>

digital pcr-based competitive index for high-throughput analysis of fitness in salmonella

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.

Watch this Scientific Journal Video about Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella at JoVE.com

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

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.

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

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 ...

digital-pcr-based-competitive-index-for-high-throughput-analysis

Nanyang Technological University

Research

JoVE Journal

Genetics

9.6K Views.  Creighton University School of Medicine. Deze techniek maakt nauwkeurige en nauwkeurige kwantificering van micro-organismen uit een gemengde populatie van zeer vergelijkbare stammen met behulp van snelle en hoge doorvoer moleculaire technieken. Uiteindelijk, in het mogelijk maken van vergelijkingen van fitness worden gemaakt tussen stammen. De mogelijkheid om tegelijkertijd veel stammen in één zwembad te beoordelen vermindert de goed-tot-welzijn of de variabiliteit van organismen aanzienlijk omdat alle stammen aan precies dezelfde...

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

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

Environment

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

Immunology and Infection

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription factors, histone modifications, and other DNA-associated proteins. ChIP-seq data can also be used to find differential binding of transcription factors in different environmental conditions or cell types. Initially, ChIP was performed through hybridization on a microarray (ChIP-chip); however, ChIP-seq has become the preferred method through technological advancements, decreasing financial barriers to sequencing, and massive amounts of high-quality data output. Techniques of performing ChIP-seq with bacterial biofilms, a major source of persistent and chronic infections, are described in this protocol. ChIP-seq is performed on Salmonella enterica serovar Typhimurium biofilm and planktonic cells, targeting the master biofilm regulator, CsgD, to determine differential binding in the two cell types. Here, we demonstrate the appropriate amount of biofilm to harvest, normalizing to a planktonic control sample, homogenizing biofilm for cross-linker access, and performing routine ChIP-seq steps to obtain high quality sequencing results.

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) is a method used to establish interactions between transcription factors and the genomic sequences they control. This protocol outlines techniques for performing ChIP-seq with bacterial biofilms, using Salmonella enterica serovar Typhimurium bacterial biofilm as an example.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Biology

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Modified Most Probable Number Assay to Quantify Salmonella in Raw and Ready-to-Cook Chicken Products

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella focus on prevalence rather than quantification, which limits their utility in assessing contamination levels and risks. This study introduces a novel most probable number (MPN) assay designed to quantify Salmonella in ready-to-cook poultry products, such as chicken cordon bleu. The method involves washing the poultry sample, concentrating the rinse through centrifugation, and serially diluting it in a 48-well block. The MPN assay is integrated with the loop-mediated isothermal amplification (LAMP) method to provide a sensitive, accurate, and rapid quantification of Salmonella contamination within the same timeframe as existing Food Safety and Inspection Service (FSIS) protocols. Results show a strong linear correlation between the MPN-LAMP measurements and theoretical inoculation levels (R&#178; = 0.933). However, variability at lower concentrations highlights challenges in accurately detecting Salmonella at these levels, with the practical lower limit of detection estimated at approximately 300 CFU/g. Potential refinements to improve the protocol's applicability include increasing the quantity sampled to further improve the limit of detection, optimizing enrichment media formulations, and expanding molecular detection to target multiple Salmonella serovars. Overall, this study presents a practical tool for the food industry, enabling reliable quantification of Salmonella contamination in poultry products, contributing to improved food safety and public health.

Accurately quantifying Salmonella in poultry at low levels is a current industrial and regulatory challenge. This protocol describes an MPN assay that enables quantification of Salmonella in raw and ready-to-cook poultry products. This method is fast, sensitive, and aligns with FSIS guidelines, enhancing food safety and supporting public health efforts.

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella ...

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

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