Research in the lab was supported by Grants from the Deutsche Forschungsgemeinschaft (DFG HA 3125/3-2, DFG HA 3125/4-2) and the Federal Ministry of Education and Research (BMBF) Medical Infection Genomics (FKZ 0315828A) to SH.

The animal infection experiments described here must be performed in strict accordance with the local and international (e.g. European Health Law of the Federation of Laboratory Animal Science Associations (FELASA)) guidelines and regulations for the use of vertebrate animals. The experiments have to be approved by the local ethical board and Institutional Animal Care Committee. All experiments with S. pneumoniae in the laboratory or the animal infections are conducted in a Class II Biosafety Cabinet.
1. Preparation of the Infectious Stock of Bioluminescent Pneumocococi
<ol>
	<li>Prepare stock pneumococcal cultures in Todd-Hewitt broth supplemented with 1% (w/v) yeast extract and a final concentration of 20% glycerol for maintenance at 80 &#176;C.</li>
	<li>Plate a small amount of deep-frozen stock pneumococcal culture onto a blood agar plate and incubate the bacteria for 10 hr at 37 &#176;C in 5% CO2. The blood agar plate contains the appropriate antibiotics such as kanamycin (150 &#181;g/ml) for the transposon insertion of the luxABCDE gene cassette into the genome.</li>
	<li>Sub-cultivate a fresh colony onto a new blood agar plate for a maximum of 10 hr.</li>
	<li>Inoculate THY medium supplemented with 10% (v/v) heat inactivated (30 min at 56 &#176;C) fetal bovine serum (FBS) with pneumococci and start the culture at an OD600 of 0.05-0.07.</li>
	<li>Incubate pneumococci without agitation at 37 &#176;C and 5% CO2 until the culture reaches an OD600 of 0.35-0.40. Growth will take between 3-4 hr.</li>
	<li>Harvest pneumococci by centrifugation at 3,750 x g for 10 min and resuspend bioluminescent pneumococci in phosphate buffered saline pH 7.4 (PBS) supplemented with 0.5% FBS.</li>
	<li>Adjust the inoculum to the desired concentration in PBS/10% FBS. The infection dose depends on the pneumococcal strain used and the genetic background of the mice. To infect outbred CD-1 mice intranasally with S. pneumoniae D39lux the infection dose should be adjusted to 1 x 107 CFU in a suspension of 20 &#181;l supplemented with 90 Units hyaluronidase. Do not shake or vortex pneumococci, since this will trigger autolysis.</li>
	<li>Verify the inoculum concentration by plating 10&#160;fold serial dilutions onto blood agar and enumeration of the colonies after overnight growth at 37 &#176;C in 5% CO2.</li>
	<li>Verify the bioluminescence of the pneumococcal inoculum by measuring the bioluminescence using an optical imaging system.</li>
</ol>
2. Intranasal and Intraperitoneal Infection of Mice with Bioluminescent Pneumococci
<ol>
	<li>Use female outbred mice in the age of 7-10 weeks for the acute pneumonia and sepsis model. The weight of these mice should be between 20-30 g.</li>
	<li>Anesthetize mice by intraperitoneal injection of ketamine and xylazine. Prepare a mixture 1.0 mg ketamine and 0.1 mg xylazine in 100 &#181;l sterile 0.9 % (w/v) sodium chloride for mice with a body weight of 20 g. This is consistent with&#160;a dose of 50 mg/kg body weight for ketamine and 5 mg/kg body weight for xylazine. To be accurate, weigh mice before the injection of the anesthetics.</li>
	<li>Inject the mixture into the intraperitoneal cavity and place the animals back into the cage until the anesthetics act and the animals are narcotized. Breathing of mice will become very slow and regular. Mice have to be fully narcotized, so that the intranasal inoculation and inhalation of the bacterial suspension will not result in sneezing and hence, loss of inoculum. This can be assessed by pinching the mouse at the end of their tail; a fully narcotized mouse will lack responsiveness.</li>
	<li>Gently pick up a narcotized mouse and hold the mouse between the fingers and thumb with the nose upright (Figure 1).</li>
	<li>Use a pipette with long narrow tips (Gel Loader Tips) and drop bioluminescent pneumococci in multiple small droplets onto the nares (10 &#181;l/nostril) of the mouse26-29. The mouse will involuntarily inhale the bacteria. Hold the mouse 1-2 min upright and observe whether the mouse sneezes or keeps the inoculum.</li>
	<li>Infect 7-10 week old CD-1 mice intranasally with an infection dose of 1 x 107&#160;or 7.5 x 107&#160;pneumococci of strain D39 and TIGR4, respectively, to monitor the infection in real&#160;time. The detection limit for the system without any absorption by the host tissue is approximately 1 x 106 bioluminescent bacteria.</li>
	<li>Infect mice intraperitoneally (I.P.) to assess and bioimage virulence of pneumococci in a sepsis mouse infection model. Use 5 x 103 CFU in 100 &#181;l of strain D39 and 1 x 104 of strain TIGR4. Mice are not anesthetized when using the I.P. route.</li>
	<li>Inoculate control mice with PBS. 
	<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51174/51174fig1highres.jpg" src="/files/ftp_upload/51174/51174fig1.jpg" width="500px" /> 
	Figure 1. Intranasal infection of CD-1 mice with S. pneumoniae. <a href="https://www.jove.com/files/ftp_upload/51174/51174fig1highres.jpg" target="_blank">Click here to view larger image</a>.</li>
</ol>
3. Visualizing Pneumococcal Dissemination into the Lung and Blood Using the Imaging System
Image dissemination of pneumococci after intranasal infection of female outbred CD1 mice in real-time using an in vivo imaging system.
<ol>
	<li>Switch on the imaging system and start the image analysis software located on the preconfigurated computer.</li>
	<li>The imaging system initializes automatically and the CCD camera is thermoelectrically cooled to 90 &#176;C before being active.</li>
	<li>The settings and binning depend on the number of mice measured simultaneously and on the strength of the bioluminescent signal. The field of view (FOV) to monitor the bioluminescence of the animals is variable and the magnification ranges between an FOV of 22.5 cm to measure five mice, and an FOV of 3.9 cm to measure a single mouse or parts of the animal like the chest or head.</li>
	<li>Use routinely an exposure time of 1 min, a medium degree of binning, and an FOV of 22.5 cm (set D). Firstly, select the luminescent&#160;imaging mode and then set the imaging parameters.</li>
	<li>The emission of the signal may depend on the bacterial strain and mouse strain used, and the pigment of the mouse. Shave the chest of mice when using e.g. C57BL/6 mice, which can be advantageous when imaging the development of pneumonia in these mice.</li>
	<li>Image infected mice at prechosen time intervals. Measure the bioluminescence of mice in the acute pneumonia model at time intervals of maximum 8-12 hr. Monitor also the welfare of infected mice by observing their appearance, behavior, and determination of the loss of weight.</li>
	<li>Anesthetize mice in the narcotic chamber of the anesthesia system of the imaging equipment prior to the measurements in the chamber. Start inhalation of a mixture of isoflurane and oxygen and wait until&#160;the mice breathe slowly and regularly.</li>
	<li>Place anesthetized animals into the imaging chamber of the system and keep mice in the supine position. The CCD camera on the top of the chamber will then image the murine respiratory tract.</li>
	<li>Place the animals with their nose into to tubes to allow inhalation of the anesthetics.</li>
	<li>Isoflurane entry into the imaging chamber via a gas tubing is allowed to maintain anesthesia.</li>
	<li>To activate the CCD camera on top of the chamber and to start bioluminescent optical imaging, press &#8220;Acquire&#8221; in the imaging software. Immediately a photograph of the mice in the closed chamber appears on the screen. After one minute of measurement an overlay of the bioluminescence data and the photo is shown.</li>
	<li>Stop anesthesia after the image overlay appears in the window of the software and put the mice back into their cages.</li>
	<li>Mice are monitored for recovery. 
	<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51174/51174fig2highres.jpg" src="/files/ftp_upload/51174/51174fig2.jpg" width="500px" /> 
	Figure 2. Anesthesia and monitoring of mice infected with bioluminescent pneumococci using the IVIS anesthesia and IVIS Spectrum system, respectively.&#160;A) The IVIS Spectrum imaging system. B) The IVIS anesthesia system with the incubation chamber. C) Mice anesthetized in the incubation chamber. D) Mice located within the IVIS Spectrum Imaging chamber with their nose inhaling the anesthetic. <a href="https://www.jove.com/files/ftp_upload/51174/51174fig2highres.jpg" target="_blank">Click here to view larger image</a>.</li>
</ol>

4. Quantification and Evaluation of the Bioluminescence of Mice Infected with Bioluminescent S. pneumoniae
<ol>
	<li>Determine the bioluminescence intensities of the mice or of a selected region of the mice by quantifying the total photon emission (photons/sec) using the image analysis software.</li>
	<li>Use the values of the photon emission that are provided in an Excel data sheet to prepare a graph. Since the data variation is often high within the grouped mice, generate a box whisker graph to display the differences in mice infected with the different pneumococcal strains.</li>
	<li>Provide the photons per mice or alternatively the results as the average of radiance (photons/sec/cm2/sr) of a selected area.</li>
	<li>Measurement data of ROI (region-of-interest) in photon mode can be quantitatively compared across different in vivo imaging systems with different camera settings, since the measurements in units of radiance automatically take into account camera settings (e.g. integration time, binning, f/stop, and field of view).</li>
</ol>

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The authors have nothing to disclose.

All experiments conducted in animals have to be approved by local authorities and ethics commissions. In in vivo infection experiments the bacterial load in the various host niches of infected animals has to be determined at various time points post-infection. Under these experimental conditions the animals have to be sacrificed prior to the isolation of the bacteria from the blood, nasopharynx, bronchoalvelar lavage, or organs such as the lungs, spleen, and brain. To calculate the number of bacteria per host ni...

Respiratory tract infections caused by viruses or bacteria remain one of the most common community-acquired or clinical problems worldwide causing approximately one third of all death worldwide. The key bacterial species are Haemophilus influenzae and Streptococcus pneumoniae2.&#160;However, these bacterial species are normally common constituents of the natural respiratory tract flora. Thus bacterial carriage is also of certain risk for invasive disease and depending on the immune status or predispositions of the individuals. The asymptomatic colonization is triggered to invasive infections. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. In healthy individuals S. pneumoniae (pneumococci) are often asymptomatic and harmless colonizers of the upper respiratory tract, where they are confronted with nonpathogenic bacteria of the resident flora but also with pathogens such as Haemophilus spp. or Staphylococcus aureus&#160;and the first line of the human immune defense system. Carriage rates are highest in young children (37%) and even higher within crowded day care centers (58%)3-5. The youngest population and the elderly, receiving the pneumococcus via aerosol transmission from carriers and nasopharyngeal secretions6, belong to the high risk groups and vaccination using one of the pneumococcal conjugate vaccines (PCV10 or PCV13 in children and 23-valent polysaccharide PPSV23 in adults) is recommended in the United States (US) and many European countries4. The PPSV23 covers serotypes responsible for ~90% of the bacteremic pneumococcal diseases in the US and Europe, preventing thus efficiently invasive pneumococcal diseases (IPD) in adults, while the PCVs cover the most prevalent serotypes in children. Consequently, IPD due to vaccine types (VT) are reduced but nonvaccine serotypes displaying a high virulence potential and antibiotic resistance have emerged4,7-12. The nasopharynx as the reservoir is the starting point for pneumococci to spread to the sinuses or middle ears initiating harmful local infections. More important, pneumococci spread directly via the airway to the bronchia and lung resulting in life-threatening CAP4,13. Lung infections are often accompanied with tissue and barrier destruction, thus enabling the pathogen to spread into the blood and causing IPD. Incidences of CAP and IPD are highest in immunocompromised persons or at the extremes of age4,13. The circumstances responsible for the conversion from a commensal to a pathogen with high virulence are still under debate. However, besides changes in the host susceptibility and evolutionary adaptation accompanied with higher virulence and the increase in antibiotic resistances have been suggested to have a crucial impact on pneumococcal infections14-16.
The pathogen is endowed with a multiplicity of adhesins mediating intimate contact to mucosal epithelial cells. After surmounting the airway mucus, pneumococcal adherence to host cells is facilitated via direct interactions of surface-exposed adhesins with cellular receptors and by exploiting extracellular matrix components or serum proteins as bridging molecules4,17,18. As versatile pathogens pneumococci are also equipped with factors involved in evasion of host immune defense mechanisms. Moreover, they have the capacity to adapt to various host milieus such as the lung, blood, and cerebrospinal fluid (CSF), respectively5,17,19,20.
The impact of bacterial factors on pathogenesis and inflammatory host responses is investigated in experimental animal models of pneumonia, bacteremia, or meningitis21-25. Despite being a human pathogen, these models are well-established to decipher pneumococcal tissue tropism, virulence mechanisms, or protectivity of pneumococcal vaccine candidates. The genetic background of inbred mouse strains determines the susceptibility to pneumococci. BALB/c mice intranasally infected with pneumococci were found to be resistant, while CBA/Ca and SJL mice were more susceptible against pneumococcal infections22. This implies that, similar to humans, the genetic background and the host defense mechanisms determine&#160;the outcome of the infection. Hence, further efforts are necessary to unravel resistance loci in the genome of mice less susceptible to pneumococcal infections. The findings have led to changes in in vivo virulence protocols. Instead of the inbred BALB/c mice often used in the past, the highly susceptible CD-1/MF1 outbred mouse strains are nowadays often used to study the effect of loss-of-function pneumococcal virulence or fitness factors26-28. Moreover, the availability of bioluminescent pneumococci and optical imaging techniques allows the real-time bioluminescence bioimaging of infections. In pneumococci the optimized luxABCDE gene cassette (plasmid pAUL-A Tn4001 luxABCDE Kmr) has been inserted into a single integration site of the chromosome by transposon mutagenesis. Bioluminescent pneumococci have been employed to assess the attenuation of pneumococcal mutants deficient in virulence or fitness factors and their translocation from one anatomical site to another26,28-31.
Here we provide a protocol for the bioimaging of pneumococcal infections in a murine pneumonia or sepsis model. Amplification and dissemination of bioluminescent pneumococci in intranasally or intraperitoneally infected mice can easily be monitored over time using an optical imaging system and the same animal at different time points.

<table border="0" cellpadding="0" cellspacing="0" width="985">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Todd Hewitt broth</td>
			<td style="width:280px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:129px;">X936.1</td>
			<td style="width:280px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td style="width:280px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:129px;">2363.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Blood agar plates</td>
			<td style="width:280px;">Oxoid, Wesel, Germany</td>
			<td style="width:129px;">PB5039A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kanamycin</td>
			<td style="width:280px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:129px;">T832.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erythromycin</td>
			<td style="width:280px;">Sigma-Aldrich,Taufkirchen, Germany</td>
			<td style="width:129px;">E6376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">fetal bovine serum (FBS)</td>
			<td style="width:280px;">PAA Laboratories, Coelbe, Germany</td>
			<td style="width:129px;">A11-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">CD-1 mice, female</td>
			<td style="width:280px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:129px;">CD1SIFE06W08W</td>
			<td>female CD-1 mice, six to eight weeks old</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamin 500mg, Curamed injection solution</td>
			<td>Schwabe-Curamed, Karlsruhe, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Rompun 2%, injection solution</td>
			<td>Bayer Animal Health, Monheim, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Plastipak 1 ml syringes</td>
			<td>Becton Dickinson, Heidelberg, Germany</td>
			<td style="width:129px;">300015</td>
			<td>sterile Luer-Lok&#8482; syringes with needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Gel Loader Tips</td>
			<td style="width:280px;">peqlab</td>
			<td style="width:129px;">81-13790</td>
			<td>M&#181;ltiFlex&#8482; Tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Hyaluronidase</td>
			<td style="width:280px;">Sigma-Aldrich</td>
			<td style="width:129px;">H3884-100mg</td>
			<td>Hyaluronidase Type IV-S from Bovine test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Oxygen</td>
			<td style="width:280px;">Air Liquide, D&#252;sseldorf, Germany</td>
			<td style="width:129px;">M1001L50R2A001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Isofluoran</td>
			<td style="width:280px;">Baxter, Unterschlei&#223;heim, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pGEM-T Easy</td>
			<td style="width:280px;">Promega, Mannheim, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Oligonucleotides </td>
			<td style="width:280px;">Eurofins MWG, Ebersberg, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Qiaprep Spin Midiprep Kit </td>
			<td style="width:280px;">Qiagen, Hilden, Germany</td>
			<td style="width:129px;">27104</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:296px;">PCR DNA purification kit</td>
			<td style="width:280px;">Qiagen, Hilden, Germany</td>
			<td style="width:129px;">28106</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:296px;">Equipment</td>
			<td style="width:280px;"> </td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Living Image 4.1 software</td>
			<td style="width:280px;">Caliper Life Sciences/PerkinElmer, Rodgau, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">XGI-8 Gas Anesthesia System</td>
			<td style="width:280px;">Caliper Life Sciences/PerkinElmer, Rodgau, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">IVIS Spectrum Imaging System </td>
			<td style="width:280px;">Caliper Life Sciences/PerkinElmer, Rodgau, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Biophotometer</td>
			<td style="width:280px;">Eppendorf AG, Hamburg, Germany</td>
			<td style="width:129px;"> </td>
			<td></td>
		</tr>
	</tbody>
</table>

following in real time the impact of pneumococcal virulence factors in an acute mouse pneumonia model using bioluminescent bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

The acquisition and uptake of methionine is of central importance for pneumococci to maintain fitness in their host niche32,33. The methionine ABC transporter lipoprotein is encoded in D39 by the spd_0151 gene (TIGR4: sp_0149) and named MetQ 32. Pneumococci further produce methionine biosynthesis enzymes (D39: Spd_0510 &#8211; Spd_0511; TIGR4 Sp_0585 &#8211; Sp_0586, MetE and MetF). The lack of methionine in a chemically defined medium affects growth of pneumococci and sim...

Watch this Scientific Journal Video about Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria at JoVE.com

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

following-real-time-impact-pneumococcal-virulence-factors-an-acute

Research

JoVE Journal

Immunology and Infection

14.9K Views.  University of Greifswald. Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2 million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Video: Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...