This work was supported by grants from the National Key Research and Development Program of China [2017YFD0500102]; the National Natural Science Foundation of China [31572544]; the State Key Laboratory of Veterinary Etiological Biology [SKLVEB2016KFKT005]; the Shanghai Agriculture Applied Technology Development Program, China [G2016060201].

The mouse infection experiments were approved by the Laboratory Animal Monitoring Committee of Jiangsu Province, China and performed in the Laboratory Animal Center of Nanjing Agricultural University (Permit number: SYXK (Su) 2017-0007).
1. Preparation of Bacteria
Note: S. suis serotype 2 virulent strain P1/7 was isolated from a diseased pig with meningitis19. Strain P1/7 was grown in Todd-Hewitt broth (THB, formula per liter of THB: H...

<ol>
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P., Xu, J., Segura, M., Gottschalk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+suis,+an+important+pig+pathogen+and+emerging+zoonotic+agent-an+update+on+the+worldwide+distribution+based+on+serotyping+and+sequence+typing.">Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing.</a> Emerging Microbes &amp; Infections. 3 (6), e45 (2014).</li><li>Kerdsin, 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=Emergence+of+Streptococcus+suis+serotype+9+infection+in+humans.">Emergence of Streptococcus suis serotype 9 infection in humans.</a> Journal of Microbiology, Immunology and Infection. 50 (4), 545-546 (2017).</li><li>Hatrongjit, 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=First+human+case+report+of+sepsis+due+to+infection+with+Streptococcus+suis+serotype+31+in+Thailand.">First human case report of sepsis due to infection with Streptococcus suis serotype 31 in Thailand.</a> BMC Infect Diseases. 15, 392 (2015).</li><li>Fittipaldi, N., Segura, M., Grenier, D., Gottschalk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+factors+involved+in+the+pathogenesis+of+the+infection+caused+by+the+swine+pathogen+and+zoonotic+agent+Streptococcus+suis.">Virulence factors involved in the pathogenesis of the infection caused by the swine pathogen and zoonotic agent Streptococcus suis.</a> Future Microbiology. 7 (2), 259-279 (2012).</li><li>Hall-Stoodley, L., Costerton, J. 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The intracranial subarachnoidal route of infection described here has obvious advantages over other routes of infection. It allows investigators to study the host-bacterium interaction and the effect of bacterial components on host immune responses directly in the brain, which mimic bacterial entrance into the central nervous system. Thus, this route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against ...

Streptococcus suis (S. suis) is a major bacterial pathogen of pigs worldwide, causing severe diseases including meningitis, pneumonia, septicaemia, endocarditis, and arthritis1. It is also an emerging zoonotic agent. So far, it has been reported that nine serotypes can cause infection in humans, including serotypes 2, 4, 5, 9, 14, 16, 21, 24, and 312,3,4. In humans and pigs, meningitis is one of the major clinical signs of S. suis infections. In Vietnam and Thailand, S. suis is the major cause of meningitis in adults5. Microbial biofilms are microorganisms that adhere to each other and are concentrated at an interface; they are essential for bacterial virulence, survival in diverse environments, and antibiotic resistance5. Biofilms are typically surrounded by an extracellular matrix that generally contains polysaccharides, proteins, and DNA6. The latter is able to elicit host inflammatory responses and cytokine production7. Biofilm formation has been reported to be involved in streptococcal meningitis in previous studies. Biofilms contribute to Streptococcus agalactiae meningitis in a tilapia fish model and biofilm formation has been revealed within brain tissues and around meningeal surfaces in vivo through intra-abdominal inoculation8. During meningitis, Streptococcus pneumoniae is in a biofilm-like state and bacteria in such a biofilm state were more effective in inducing meningitis in a mouse infection model9. In addition, in our previous study, the biofilm state associated with S. suis in mouse brain contributes to bacterial virulence by survival analysis10. However, direct evidence for biofilm involvement in S. suis meningitis requires further investigation.
Animal models of S. suis infection have been developed in mice using the intraperitoneal (i.p.)11, intranasal (i.n.)12, intravenous (i.v.)13, and the intracisternal (i.c.) routes of infection14,15,16. However, the i.p., i.n., and i.v. routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain. These include extracellular matrix from biofilms. Although the i.c. inoculation was used for S. suis infection, the precise injection site has not been described in those papers. In contrast, the stereotaxic coordinates of the injection site for intracranial subarachnoidal inoculation has clearly been described in a previous study17. This allowed easy recognition of the inoculation point and more simplistic experimental protocol. In addition, the intracranial subarachnoidal route of infection mimics bacterial entrance into the central nervous system from the sinuses or the middle ear17, and the relationship between the middle ear and meningitis caused by S. suis has been demonstrated by Madsen et al18. Moreover, by applying the intracranial subarachnoidal route of infection in mice, we have demonstrated that S. suis small RNA rss04 contributes to meningitis in our previous study10.
In the present study, the intracranial subarachnoidal route of infection was used in mice to investigate the roles of biofilms in S. suis meningitis. Mice were infected with planktonic cells or biofilm state cells of S. suis by this route of infection. Histopathological analysis and increased mRNA expression of TLR2 and cytokines from brain tissue of mice injected with biofilm state cells clearly indicated that S. suis biofilm contributes to meningitis.

<table border="0" cellpadding="0" cellspacing="0" style="width:828px;" width="827">
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		<col />
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		<col />
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	<tbody>
		<tr>
			<td height="46">Todd Hewitt Broth(THB)</td>
			<td style="width:225px;">Becton, Dickinson and Company</td>
			<td style="width:115px;">DF0492078</td>
			<td style="width:304px;">Dissolve 30 g of the powder in 1 L of purified water. Autoclave at 121&#176; for 15 min.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td style="width:225px;">DSBIO</td>
			<td style="width:115px;">16C0050</td>
			<td style="width:304px;">Dissolve 15 g of the powder in 1 L of THB. Autoclave at 121&#176; for 15 min.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:184px;">Milli-Q Reference Water Purification System</td>
			<td style="width:225px;">Merck KGaA</td>
			<td style="width:115px;">Z00QSVCUS</td>
			<td style="width:304px;">Without Dnase/ Rnase</td>
		</tr>
		<tr height="21">
			<td>NaCl</td>
			<td style="width:225px;">Tianjin Kemiou Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10019318</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="21">
			<td>Na2HPO3</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009012-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="21">
			<td>KCl</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009017-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="20">
			<td>KH2PO4</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009019-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="24">
			<td>KOH</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009014-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="42">
			<td>Glycerol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10010618</td>
			<td style="width:304px;">Diluted with equal volumu of purified water, autoclave at 121&#176; for 15 min</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">4% paraformaldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">80096675</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">25% Glutaraldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">30092436</td>
			<td style="width:304px;">10-fold diluted with purified water for fixation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Ethanol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10009218</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10006818</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spctrophotometre</td>
			<td style="width:225px;">DeNovix Inc.</td>
			<td style="width:115px;">DS-11+</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Ultrasound cell crusher</td>
			<td style="width:225px;">NingBo Scientz Biotechnology Co.,Ltd</td>
			<td style="width:115px;">JY96-IIN</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Centrifuge</td>
			<td style="width:225px;">Hitachi Koki Co., Ltd</td>
			<td style="width:115px;">CT15RE</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Refrigerator</td>
			<td style="width:225px;">Aucma Co., Ltd</td>
			<td style="width:115px;">DW-86L500</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Scanning electron microscope</td>
			<td>Zeiss</td>
			<td style="width:115px;">EVO-LS10</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastRNA Pro Green Kit</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">#6045-050</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPrep-24 Instrument</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">116005500</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Instrument for PCR</td>
			<td style="width:225px;">SensoQuest GmbH</td>
			<td style="width:115px;">1124310110</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuantStudio 6 Flex</td>
			<td style="width:225px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">4485689</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">SYBR Premix Ex Taq II</td>
			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR820A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">PrimeScript RT reagent kit with gDNA Eraser</td>
			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR047A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Fully Enclosed Tissue Processor</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ASP200S</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Heated Paraffin Embedding Module</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">EG1150H</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Semi-Automated Rotary Microtome</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">RM2245</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Water bath for paraffin sections</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">HI1210</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Autostainer XL</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ST5010</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Agilent 2100</td>
			<td style="width:225px;">Agilent Technologies</td>
			<td style="width:115px;">G2939A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Optical microscope</td>
			<td style="width:225px;">Olympus</td>
			<td style="width:115px;">BX51</td>
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intracranial subarachnoidal route of infection for investigating roles of streptococcus suis biofilms in meningitis in a mouse infection model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

SEM analysis was performed to examine biofilm formation under the experimental conditions. As shown in Figure 1, there is a significant difference in biofilm formation between planktonic cells (Figure 1A) and biofilm state cells (Figure 1B). SEM analysis showed that biofilm bacteria were in clumps and multiple layers and they were encased in the extracellular matrix,...

Watch this Scientific Journal Video about Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model at JoVE.com

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

This method can help answer key questions about the roles of streptococcus suis'components in many sites, such as the extracellular matrix from its biofilms. The main advantage of this technique is that it enables investigations of bacterial components that affect how the immune responses directly in a brain and mimic bacteria entrance into the central nervous system. Demonstrating the procedure will be Mister Shouming Zhang, a graduate student from my laboratory.To collect strain P1/7 planktonic cells, first collect five milliliters of the cells from mid-lock phase culture. Centrifuge for three minutes at 8000 times G before washing the cells three times in PBS. Re-suspend the cells with five milliliters of 25%glycerol in THB and aliquot into five tubes.Alternatively, to collect strain P1/7 biofilm state cells, take 20 milliliters of an overnight culture and add to 180 milliliters of fresh THB. Equally divide the diluted culture into 10 round culture plates, then incubate the plates at 37 degrees celsius in 5%carbon dioxide for 24 hours. Following incubation, shake the plate gently to re-suspend the bacteria that have not adhered to the plate and avoid re-suspending the sediment.Then, discard the supernatant by aspiration. Add five milliliters of PBS to harvest biofilm state cells. Re-suspend the sediment completely and then transfer the sample to a new tube.Sonicate the re-suspended biofilm state cells as listed in the text protocol. Next, centrifuge the sonicated cells for three minutes at 8000 times G and store the supernatant at minus 80 degrees celsius. The supernatant may contain biofilm components and it can be used to re-suspend the biofilm bacteria before infection.Re-suspend the sediment with 10 milliliters of 25%glycerol in THB before observing the samples by scanning electron microscopy as described in the text protocol. Infect all mice using planktonic cells or biofilm state cells and perform euthanasia as described in the text protocol. Next, extract the total RNA from brain tissue using an RNA extraction kit.For each mouse, use whole brain tissue for extracting RNA. Divide whole brain tissue into five tubes. Take up to 100 milligrams of brain tissue and add one milliliter of lysis solution to each tube containing the lysis matrix provided by the kit.Extract the RNA following the steps listed in the text protocol. Next, perform CDMA synthesis, including elimination of genomic DNA, using a thermocycler with a reverse transcription reagent kit. Add one microgram of RNA to a tube containing two microliters of 5X genomic DNA elimination buffer and one microliter of geneomic DNA elimination enzyme.Add RNAse free water until the volume reaches 10 microliters. Incubate for two minutes at 42 degrees celsius. Add 10 microliters of master mix to the reaction solution and then mix gently.Proceed immediately with the reverse transcription reaction by placing the tube at 37 degrees celsius for 15 minutes, then transfer the reaction to 85 degrees celsius for five seconds. Perform the quantitative real-time PCR analysis using a real-time PCR machine with a cyber quantitative real-time PCR kit. Following the kit instructions, combine the enzyme, primers, 50X ROX reference dye two, and CDMA template, then add RNAse free water until a total volume of 20 microliters is reached.Run each sample in triplicate using the thermal parameters listed in the text protocol and calculate the relative fold-change based on the two minus delta delta CT method. Also perform the observation and scoring of histological sections of the infected brain tissue as detailed in the text protocol. The effect of biofilms on inflammatory response in mirroring brain tissue is shown here.At 12 hours post-infection, the expression of CCL2, IL6, TNF-alpha, and TLR2 from brains of infected mice was significantly higher for biofilm state cells as compared to planktonic cells. Far more severe pathological changes were observed in the meninges and cerebral cortex of mice infected with the biofilm bacteria as compared to mice infected with the planktonic bacteria. These changes included large areas of necrosis and intense inflammatory cell infiltration.However, much less inflammatory cell infiltration was observed in the meninges from mice infected with the planktonic bacteria. Furthermore, there was far more intense inflammatory cell infiltration observed in ventricles from mice infected with biofilm bacteria as compared to mice infected with the planktonic bacteria. As shown here, much less inflammatory cell infiltration was observed in ventricles from mice infected with the planktonic bacteria.While attempting this procedure, it is important to keep in mind that different strains may have different infective doses, so a preliminary experiment is strongly recommended to determine the appropriate infection dose. After watching this video, you should have a good understanding of how to collect biofilm cells and perform intercranial subretinal direct infection in mice.

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JoVE Journal

Immunology and Infection

7.2K visualizações.  Nanjing Agricultural University. This method can help answer key questions about the roles of streptococcus suis'components in many sites, such as the extracellular matrix from its biofilms. The main advantage of this technique is that it enables investigations of bacterial components that affect how the immune responses directly in a brain and mimic bacteria entrance into the central nervous system. Demonstrating the procedure will be Mister Shouming Zhang, a graduate student from my laboratory.To collect strain P1/7...