This work was partly supported by Takeda Science Foundation, Pfizer Academic Contribution and JSPS Postdoctoral Fellowship for Foreign Researchers (FC).

NOTE: The strains and materials used in this work are listed in Table 1 and the Table of Materials, respectively. In the transmission experiments, N315 and COL cfr-positive derivatives were used as donors of the cfr gene (N315-45 and COL-45). These strains were previously obtained by conjugation, using as the donor a clinical cfr-positive Staphyloccocus epidermidis strain (ST2), following the standard conjugation protocol (see below). This strain harbored the cfr gene on a pSCFS7-like plasmid7.
1. Conjugation Using the Filter-mating Method
NOTE: An S. aureus N315 strain carrying the cfr gene (N315-45)7 (CmR) was used as the donor. COL8 or Mu509 strains (TetR, CmS) were used as the recipients. Double-resistant colonies (TetR, CmR) able to grow in the presence of 32 mg/L of chloramphenicol plus 8 mg/L of tetracycline were considered as putative transconjugants and were analyzed to determine the presence of cfr by colony PCR and to determine the recipient susceptibility profile.
<ol>
	<li>Perform conjugation following a previously described protocol10 with a few modifications:
	<ol>
		<li>Prepare 5 mL of overnight culture of the donor and recipient in Tryptic soy broth (TSB) medium (with and without 32 mg/L chloramphenicol, respectively), with shaking at 37 &#176;C.</li>
		<li>Adjust the optical density (OD600) of the overnight cultures to 1 (OD600 = 1.0) by using fresh TSB medium. Mix 0.5 mL of the donor culture (N315-45) with 0.5 mL of the recipient culture (COL or Mu50). Add 1 ml of PBS to the mixture.</li>
		<li>Transfer the mixture of bacteria onto a 0.45 &#181;m filter membrane using a vacuum pump system.</li>
		<li>Put the filter membranes on a sheep blood agar plate. Incubate at 37 &#176;C for 1 day. 
		NOTE: In this step, enriched medium is necessary to allow the growth of double-resistant cells.</li>
		<li>Take out the filter membrane from the plate and suspend in 10 ml of phosphate-buffered saline (PBS). Vortex well to collect all the bacteria attached on the membrane.</li>
		<li>Make a serial 10-fold dilution of the bacterial suspension by using fresh TSB. Plate 100 &#181;l of the 0-10-4 diluted samples on TSB agar (TSA) plates supplemented with 32 mg/L chloramphenicol and 8 mg/L tetracycline for the selection of transconjugants. Plate 100 &#181;l of the diluted suspension (10-5-10-6) onto TSA plates with 8 mg/L tetracycline alone to count the total number of recipients. Incubate the plates at 37 &#176;C for 18-24 hr.</li>
		<li>Analyze double-resistant colonies (putative transconjugants) for the presence of the cfr gene by colony PCR7 and their susceptibility profiles (antibiogram).</li>
		<li>Determine the antibiogram by measuring the minimum inhibitory concentrations (MICs) of appropriate antibiotics according to the standard microdilution method or disk diffusion method11. The susceptibility profile of the transconjugants must be identical to the recipient, except for chloramphenicol (and other compounds affected by the cfr gene). This determination is essential to rule out tetracycline resistance developed by the donor strain.</li>
	</ol>
	</li>
</ol>
2. Phage Transduction
NOTE: The bacteriophage MR83a belonging to the Siphoviridae family (laboratory stock) was used in the transduction experiments. N315-45 was used as the cfr donor for phage infection. N315, COL, or Mu50 strains were used as the recipients. The acquisition of cfr was determined by the ability of the recipient strain to grow in the presence of 32 mg/L chloramphenicol. Colonies growing under this condition were analyzed to determine the presence of cfr (by colony PCR) and to determine the susceptibility profile to rule out potential contamination.
<ol>
	<li>Phage amplification on the donor
	<ol>
		<li>Prepare an overnight culture of N315-45 in 5 ml of nutrient broth supplemented with 3.6 mM Ca2+ (NBCaCl2) at 37 &#176;C with shaking (180 rpm). 
		NOTE: Calcium is required for phage infection. See the Materials Table. Nutrient broth from other companies might have issues, such as calcium precipitation.</li>
		<li>Prepare subcultures by diluting the overnight culture 1:1,000 in a final volume of 10 mL of NBCaCl2 in 50 ml glass flasks or glass vials. Grow the bacteria for 1 hr at 37 &#176;C with shaking (180 rpm).</li>
		<li>Prepare a series of diluted phage MR83a in NBCaCl2 medium (10-2, 10-3, 10-4, 10-5, and 10-6). Add 20 &#181;l of the diluted phage into the bacterial culture. Also prepare a control culture without phage infection, which serves as the positive control for bacterial cell growth and can be used to determine the phage titer the next day.</li>
		<li>Grow the cultures at 37 &#176;C with gentle shaking (100 rpm) overnight. 
		NOTE: The cells will grow to some extent when the infecting phage is not in excess; then, they will enter into lysis phase due to the phage amplification through the lytic cycle. The culture without phage will show full growth, while cultures with phage will show distinct rates of transparency.</li>
		<li>Select one or two cleared culture vials with the highest dilution of the added phage.</li>
		<li>Transfer the cultures into 15 ml centrifuge tubes. Add 250 &#181;l of chloroform and mix thoroughly by inverting the tubes.</li>
		<li>Centrifuge the tubes at 5,000 x g for 20 min at 4 &#176;C.</li>
		<li>Transfer the supernatants to fresh tubes and store them at 4 &#176;C until use. 
		NOTE: The phage is stable for at least a few months, but storing the phage preparation for too long reduces the titer and transduction efficiency.</li>
	</ol>
	</li>
	<li>Measuring the phage titer (phage plaque assay)
	<ol>
		<li>Prepare nutrient broth agar (1.5% agar) medium; keep it warm in a water bath at 55 &#176;C. Add autoclaved 0.5 M CaCl2 solution to the medium to a final concentration of 3.6 mM. Pour it into 90 mm Petri dishes (NBCaCl2 plates).</li>
		<li>Prepare an overnight culture of N315 in 5 ml of NBCaCl2 at 37 &#176;C with shaking; this can be substituted with the control culture described above (step 2.1.3).</li>
		<li>Add 10 &#181;l of the overnight culture into 200 &#181;l of NBCaCl2 and evenly spread it onto the NBCaCl2 plate. Stop the spreading when the surface of the plate is covered with liquid. Let the plate dry.</li>
		<li>Make a serial 1:10 dilution (from 10-5-10-10) of previously prepared phage (step 2.1.8) using NBCaCl2 medium.</li>
		<li>Spot 3 &#181;l of each phage dilution onto the plate covered with bacteria.</li>
		<li>Incubate the plate overnight at 30 &#176;C.</li>
		<li>Count the plaque numbers and calculate the phage titer using the following equation: Plaque forming unit (pfu)/ml = Number of plaques x Dilution factor/spotted volume (3 x 10-3 ml)</li>
	</ol>
	</li>
	<li>Phage transduction 
	NOTE: The phage pool prepared in the previous step (2.1.8) is used to test the transduction of the cfr gene into the S. aureus strains. In this experiment, strains N315, COL, or Mu50 are used as the recipients.
	<ol>
		<li>Prepare the overnight culture of N315, COL, or Mu50 in 5 ml of NBCaCl2 at 37 &#176;C with shaking (180 rpm).</li>
		<li>Dilute the phage in NBCaCl2 to ~109 pfu/mL.</li>
		<li>In a 50 ml glass vial, mix 500 &#181;l of overnight culture, 500 &#181;l of fresh NBCaCl2, and 1 ml of phage (~109 pfu/ml); the expected multiplicity of infection (MOI) must not exceed 1. Incubate the mixture at 37 &#176;C with gentle shaking (100 rpm) for 30 min. 
		NOTE: Heat treatment of recipient cells just prior to the addition of phage (52 &#176;C for 2 min to inactivate the endogeneous restriction enzymes) may increase the efficiency12.</li>
		<li>Add 50 &#181;l of 20% Na3-Citrate. Continue gentle shaking for 30 min at 37 &#176;C. 
		NOTE: Na3-Citrate acts as a moderate chelator of calcium.</li>
		<li>Prepare melted brain-heart infusion (BHI) agar (1.5% agar) medium and keep it in a water bath at 55 &#176;C.</li>
		<li>Transfer the bacterium-phage mixture to a 100 ml flask, add 50 ml of warm BHI agar supplemented with 32 mg/L chloramphenicol, and mix well. Pour the mixture into the 90 mm Petri dishes. 
		NOTE: This method enables the detection of a small number of growing colonies (putative transductants).</li>
		<li>Incubate the plate at 37 &#176;C for 24-48 hr.</li>
		<li>Further test the generated colonies (transductants) for resistance by transferring the colonies onto new BHI agar plates supplemented with 32 mg/L chloramphenicol. Confirm the presence of the cfr gene by colony PCR7.</li>
	</ol>
	</li>
</ol>
3. Natural Transformation
NOTE: The natural transformation assay in S. aureus was carried out following the method described in our previous study2. The N315 derivative, N2-2.12,7, was used as the recipient. In this strain, the sigH locus was duplicated so as to constitutively express SigH2. For detailed procedures on how to isolate SigH-expressing competence variants, please see a previous description2. If the resistance marker to be transferred is not chloramphenicol, pRIT-sigH (CmR) can be used to express SigH, as described previously2. Purified plasmid or whole DNA extract from cfr-acquired S. aureus COL-457 is used as the donor DNA for transformation.
<ol>
	<li>Preparation of donor DNA
	<ol>
		<li>Cultivate COL-45 overnight with shaking at 37 &#176;C in a 300 ml flask containing 50 mL of TSB supplemented with 32 mg/L chloramphenicol. Culture E. coli HST04 carrying pHY300 plasmid(TetR) to extract the plasmid for the control experiment. 
		NOTE: HST04 (dam-/dcm-) lacks the DNA methylase genes13. Other conventional strains with DNA methylases can also be used to prepare the DNA donor, because the DNA methylation state does not affect the transformation efficiency. Alternatively, pT181 plasmid purified from the S. aureus COL strain can also be used as a positive control for the transformation assay2.</li>
		<li>Collect the cells by centrifugation (8,000 x g for 10 min at 4 &#176;C).</li>
		<li>Extract the plasmids using a plasmid DNA extraction kit or a conventional DNA purification method to purify the whole DNA.</li>
		<li>Quantify the purified DNA by spectrometer and keep it at 4 &#176;C until use. 
		NOTE: Use a fresh DNA preparation for the transformation assay, usually less than one week old. Old DNA preparations reduce the efficiency of transformation.</li>
	</ol>
	</li>
	<li>Transformation assay
	<ol>
		<li>Culture the recipient cell (N2-2.1) overnight in 5 ml of TSB at 37 &#176;C with shaking.</li>
		<li>Transfer 0.5 ml of overnight culture into a 1.5 ml tube. Precipitate the cells by centrifugation (10,000 x g for 1 min at 4 &#176;C).</li>
		<li>Suspend the cells with 10 mL of CS2 medium in a 50 ml tube. 
		NOTE: CS2 medium is a complete synthetic medium that induces competence for natural transformation in S. aureus2 (see the medium composition in the Material Table). Other standard laboratory media, such as TSB or BHI, are not suitable for transformation2,13.</li>
		<li>Grow the bacteria at 37 &#176;C with shaking (180 rpm) until the late exponential phase (about 8 hr).</li>
		<li>Harvest the cells by centrifugation (5,000 x g for 5 min at 4 &#176;C).</li>
		<li>Resuspend the cells in 10 ml of fresh CS2 medium.</li>
		<li>Add 10 &#181;g of purified plasmid or genome DNA (step 3.1.4) to the cell suspension. Shake at 37 &#176;C and 180 rpm for 2.5 hr. 
		NOTE: Shorter incubation times with DNA (&#60;2.5 hr) result in lower transformation frequencies.</li>
		<li>Collect cells by centrifugation (5,000 x g for 5 min at 4 &#176;C).</li>
		<li>Resuspend the cells in 10 mL of BHI medium. Mix the cell suspension with 90 ml of melted BHI agar (55 &#176;C) supplement with 32 mg/L chloramphenicol (or 5 mg/L tetracycline in the control experiment). Pour the mixture into the 90 mm Petri dishes. Swiftly cool and let the agar solidify.</li>
		<li>Incubate the plates at 37 &#176;C for 2 days.</li>
		<li>Replicate generated colonies (transformants) by transferring the colonies (using toothpicks) to new BHI agar plates containing the appropriate antibiotics to confirm their resistance characteristics. Confirm the acquired resistance gene (cfr or tetM) by colony PCR7.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This work describes the three major methods to study the HGT of genetic determinants in S. aureus. Although transduction and conjugation have been studied for decades, the existence of natural transformation was only recently recognized2. Thus, S. aureus is equipped with all of the three major modes of HGT, and testing all of them is required to clarify the possible dissemination pathways of genetic determinants. The aim of this work is to compile complete protocols and to provid...

Staphylococcus aureus is a commensal Gram-positive bacterium that naturally inhabits the skin and nasal cavity of human beings and animals. This bacterial species is the leading cause of nosocomial infections in hospitals and healthcare settings. Moreover, its ability to develop resistance to different antimicrobial compounds has made the management of the infections caused by this bacterium into a global concern.
Two main pathways involved in the spreading of resistance phenotypes are known: the clonal dissemination of resistant genotypes and the dissemination of genetic determinants among the bacterial pool. In the case of S. aureus, different antibiotic resistance genes (as well as virulence determinants) have been found to be associated with mobile genetic elements (MGEs)1. The presence of these elements in the genome of S. aureus indicates that the acquisition and transfer of genetic material within the bacterial population could play an important role for S. aureus adaptation and evolution.
Genetic material can be exchanged through three well-known mechanisms of HGT in Gram-positive bacteria: transformation, conjugation, and phage transduction. Transformation involves the uptake of free DNA. To acquire foreign DNA, bacterial cells need to develop a special physiological phase: the competence stage. When this stage is reached, competent cells are capable of transporting DNA into the cytoplasm, acquiring new genetic determinants. In the case of S. aureus, the existence of natural transformation has been recently demonstrated2. In line with this, our group has shed light on the relevance of the expression of the SigH factor (a cryptic secondary transcription sigma factor) in the competence stage of development and on how its constitutive expression renders S. aureus capable of reaching the competence stage, which allows for the acquisition of resistant phenotypes by natural transformation2.
Conjugation is a process involving the transmission of DNA from one living cell (donor) to another (recipient). Both cells must be in direct contact, allowing the DNA to be exchanged while being protected by special structures, such as tubes or pores. The transfer of DNA by this method requires the conjugative machinery. In S. aureus, the prototype conjugative plasmid is PGO1, which harbors the conjugative operon TraA3.
Phage transduction involves the transfer of DNA from cell to cell through bacteriophage infection and implies the packing of bacterial DNA, instead of viral DNA, into the phage capsid. Most of S. aureus isolates are lysogenized by bacteriophages1. Upon stress conditions, prophages can be excised from the bacterial genome and shift to the lytic cycle.
These are the three well known mechanisms for DNA transmission in S. aureus. There are some additional transfer mechanisms, such as &#34;pseudo-transformation&#34;2 and phage-like systems in the transfer of pathogenicity islands4. Recently, one group reported that &#34;nanotubes&#34; are involved in the transfer of cellular materials (including plasmid DNA) between neighboring cells5,6, but a follow-up study has not appeared from other groups so far.
This work provides the necessary methodology to study HGT in S. aureus by addressing the three main transfer pathways: conjugation, transduction, and natural transformation. The results obtained with these methodologies were used to study the transmission of the cfr gene (chloramphenicol/florfenicol resistance) among S. aureus strains7. These three techniques are versatile tools for the investigation of MGE transmission in S. aureus.

<table><tbody><tr><td>Tryptic Soy Broth (TSB)&amp;nbsp;</td><td>Becton Dickinson&amp;nbsp;</td><td>211825</td><td></td></tr><tr><td>Brain Heart Infusion (BHI)</td><td>Becton Dickinson&amp;nbsp;</td><td>211059</td><td></td></tr><tr><td>Nutrient Broth No. 2</td><td>Oxoid</td><td>CM0067</td><td></td></tr><tr><td>Sheep blood agar</td><td>Eiken Chemical Co.,Ltd.</td><td>E-MR96</td><td>Tryptic soy agar added with 5% (v/v) sheep blood according to the manufacturer.&amp;nbsp;</td></tr><tr><td>Agar powder</td><td>Wako Pure Chemical Industries</td><td>010-08725</td><td></td></tr><tr><td>Sodium citrate (Trisodium citrate dihydrate)</td><td>Wako Pure Chemical Industries</td><td>191-01785</td><td></td></tr><tr><td>Cellulose Ester Gridded 0.45 &amp;mu;L HAWG filter</td><td>Merck Milipore</td><td>HAWG 02500</td><td></td></tr><tr><td>QIAfilter Plasmid Midi kit</td><td>QIAGEN</td><td>12243</td><td></td></tr></tbody></table>

methodology for the study of horizontal gene transfer in staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

The results represented here have been previously published (adapted from reference7 with the publisher&#39;s permission). We studied the potential transmission pathways of the cfr gene, which causes low-level linezolid resistance and the expression of the PhLOPSA-resistance phenotype14,15 in S. aureus strains, by investigating three mechanisms of HGT.
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Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

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16.5K Views.  University of Tsukuba.  We beschrijven hier drie verschillende protocollen voor de in vitro onderzoek conjugatie, transductie en natuurlijke transformatie in Staphylococcus aureus. 

Video: Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Genetics

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Biochemistry

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Heuristic Mining of Hierarchical Genotypes and Accessory Genome Loci in Bacterial Populations

Routine and systematic use of bacterial whole-genome sequencing (WGS) is enhancing the accuracy and resolution of epidemiological investigations carried out by Public Health laboratories and regulatory agencies. Large volumes of publicly available WGS data can be used to study pathogenic populations at a large scale. Recently, a freely available computational platform called ProkEvo was published to enable reproducible, automated, and scalable hierarchical-based population genomic analyses using bacterial WGS data. This implementation of ProkEvo demonstrated the importance of combining standard genotypic mapping of populations with mining of accessory genomic content for ecological inference. In particular, the work highlighted here used ProkEvo-derived outputs for population-scaled hierarchical analyses using the R programming language. The main objective was to provide a practical guide for microbiologists, ecologists, and epidemiologists by showing how to: i) use a phylogeny-guided mapping of hierarchical genotypes; ii) assess frequency distributions of genotypes as a proxy for ecological fitness; iii) determine kinship relationships and genetic diversity using specific genotypic classifications; and iv) map lineage differentiating accessory loci. To enhance reproducibility and portability, R markdown files were used to demonstrate the entire analytical approach. The example dataset contained genomic data from 2,365 isolates of the zoonotic foodborne pathogen Salmonella Newport. Phylogeny-anchored mapping of hierarchical genotypes (Serovar -&#62; BAPS1 -&#62; ST -&#62; cgMLST) revealed the population genetic structure, highlighting sequence types (STs) as the keystone differentiating genotype. Across the three most dominant lineages, ST5 and ST118 shared a common ancestor more recently than with the highly clonal ST45 phylotype. ST-based differences were further highlighted by the distribution of accessory antimicrobial resistance (AMR) loci. Lastly, a phylogeny-anchored visualization was used to combine hierarchical genotypes and AMR content to reveal the kinship structure and lineage-specific genomic signatures. Combined, this analytical approach provides some guidelines for conducting heuristic bacterial population genomic analyses using pan-genomic information.

This analytical computational platform provides practical guidance for microbiologists, ecologists, and epidemiologists interested in bacterial population genomics. Specifically, the work presented here demonstrated how to perform: i) phylogeny-guided mapping of hierarchical genotypes; ii) frequency-based analysis of genotypes; iii) kinship and clonality analyses; iv) identification of lineage differentiating accessory loci.

Routine and systematic use of bacterial whole-genome sequencing (WGS) is enhancing the accuracy and resolution of epidemiological investigations carried ...

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Visualizing Antibiotic Persistence in Bacterial Pathogens

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to&#160;underlie clinical treatment failure.&#160;Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited.
This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus)&#160;during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize&#160;phenotypic variants, such as persisters, by time-lapse epifluorescence&#160;microscopy.

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment ...

Biology

Types of Genetic Transfer Between Organisms

Genetic transfer occurs when genetic information is passed from one organism to another. It occurs via two mechanisms: vertical gene transfer and horizontal gene transfer. Vertical gene transfer occurs when genetic information is transferred from one generation to the next, which happens much more frequently than horizontal gene transfer. Both sexual and asexual reproduction are forms of vertical gene transfer, where one or more organisms pass some or all of their genome onto their progeny. Additionally, vertical gene transfer occurs in both prokaryotic and eukaryotic species.
Horizontal gene transfer occurs when genetic information is passed to a member of the same generation and happens most frequently in prokaryotic species. While inter-species horizontal gene transfer is exceedingly rare among eukaryotes, it occurs frequently in prokaryotes. Horizontal gene transfer between different species is a significant source of genetic diversity among prokaryotes.
Most prokaryotic species reproduce asexually. While this allows for faster production of offspring, the offsprings produced possess limited genetic diversity. Horizontal gene transfer, therefore, serves a vital role in introducing genetic diversity to prokaryotes. Through horizontal gene transfer, prokaryotes can share a small fraction of their genome with other organisms, either conspecific (the same species) or allospecific (a different species), in the same generation. Many scientists posit that horizontal gene transfer and mutation are the most significant sources of genetic variation in prokaryotes. Thus, horizontal gene transfer provides some of the raw material upon which natural selection acts.&#160;&#160;
A prominent example of this is the emergence of antibiotic-resistant strains of bacteria. Genes that confer resistance to an antibiotic can be transferred between different species and strains of bacteria, giving the recipient bacteria a selective advantage, such as penicillin-resistant strains of Neisseria gonorrhoeae, which causes gonorrhea. On an even grander scale, some estimates suggest that at least 18% of E. coli&#8217;s genome was acquired via horizontal gene transfer over the course of millions of years of evolution.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

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