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.</...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+and+the+pneumococcus.">Innate immunity and the pneumococcus.</a> Microbiology. 152, 285-293 (2006).</li><li>Gerber, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+Streptococcus+pneumoniae+meningitis+mimicking+several+features+of+human+disease.">A mouse model of Streptococcus pneumoniae meningitis mimicking several features of human disease.</a> Acta Neuropathol. 101, 499-508 (2001).</li><li>Gingles, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+genetic+resistance+in+invasive+pneumococcal+infection:+identification+and+study+of+susceptibility+and+resistance+in+inbred+mouse+strains.">Role of genetic resistance in invasive pneumococcal infection: identification and study of susceptibility and resistance in inbred mouse strains.</a> Infect. Immun. 69, 426-434 (2001).</li><li>Holmes, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pavA+gene+of+Streptococcus+pneumoniae+encodes+a+fibronectin-binding+protein+that+is+essential+for+virulence.">The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence.</a> Mol. Microbiol. 41, 1395-1408 (2001).</li><li>Koedel, U., Klein, M., Pfister, H. 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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">
	<colgroup>
		<col />
		<col />
		<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 visualizações.  University of Greifswald.  Streptococcus pneumoniae é o patógeno principal causador da pneumonia adquirida na comunidade grave e responsável por mais de 2 milhões de mortes em todo o mundo. O impacto dos fatores bacterianos implicados em fitness ou virulência pode ser monitorado em tempo real em uma pneumonia ou bacteremia modelo aguda mouse usando bactérias bioluminescentes. 