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Method Article

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

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DOI:

10.3791/57658

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July 1st, 2018

 ,  ,  ,  ,  ,  , 
1College of Veterinary Medicine, Nanjing Agricultural University, 2Key Lab of Animal Bacteriology, Ministry of Agriculture, 3OIE Reference Lab for Swine Streptococcosis, 4School of Medicine, Tsinghua University, 5Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses

In This Article

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

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: Heart Infusion, 3.1 g; neopeptone, 20.0 g; dextrose, 2.0 g; sodium chloride, 2.0 g; disodium phosphate, 0.4 g; sodium carbonate, 2.5 g) and plated on Todd-Hewitt agar (THA, formula per liter of THA: Heart Infusion, 3.1 g; neopeptone, 20.0 g; dextrose, 2.0 g; sodium chloride, 2.0 g; disodium phosphate, 0.4 g; sodium carbonate, 2.5 g; agar, 15.0 g) at 37 °C and 5% CO2.

  1. Collection of strain P1/7 planktonic cells
    1. Collect 5 mL of planktonic cells from mid-log phase culture (OD600=0.6), centrifuge for 3 min at 8000 × g, and then wash 3 times with PBS.
    2. Resuspend the cells with 5 mL of 25% glycerol in THB, aliquot into 5 tubes, and then store at -80 °C.
  2. Collection of strain P1/7 biofilm state cells
    1. Take 20 mL of an overnight culture and add to 180 mL of fresh THB; divide the diluted culture into 10 round culture plates equally, and then put the plates in an incubator at 37 °C in 5% CO2 for 24 h.
    2. After incubation, shake the plate gently to resuspend the bacteria that have not adhered to the plate, and then discard the supernatant by aspiration.
      NOTE: Shake the plate gently and avoid resuspending the sediment.
    3. Add 5 mL of PBS to harvest biofilm state cells, resuspend the sediment completely, and then transfer the sample to a new tube.
    4. Sonicate the biofilm state cells from the last step with the following parameters: 60 W, 4 cycles, 5 s on and 10 s off.
    5. Centrifuge for 3 min at 8000 × g and store the supernatant at -80 °C.
      NOTE: The supernatant may contain biofilm components and it can be used to resuspend the biofilm bacteria before infection as described in the animal experiments (Step 3).
    6. Resuspend the sediment with 10 mL 25% glycerol in THB and then store the biofilm state cells at -80 °C.

2. Scanning Electron Microscopy (SEM) Analysis

  1. Fix the collected biofilm state cells and planktonic cells using 4% paraformaldehyde for 12 - 24 h at 4 °C.
  2. Rinse the samples quickly with distilled water and dehydrate the samples sequentially with an increasing concentration of ethanol (25%, 50%, 70%, 90%, and 100%) for 30 min in each solution.
  3. Adhere the dried samples to metal holders with double-sided tape and finally coat them in an evaporator with gold and palladium.
  4. Observe the samples by a scanning electron microscope using the following parameters: EHT (Extra High Tension) = 10 kV; WD (Working Distance) = 7.0 or 8.0 mm; Magnification = 5000 ×; Signal A= SE1.

3. Animal Experiments

  1. Use SPF 6-week-old female BALB/c mice in this study. Infect all mice through the intracranial subarachnoidal route of infection whose injection site is located 3.5 mm rostral from the bregma. The stereotaxic coordinates of the injection site were clearly described by Chiavolini et al17.
    1. For histopathological analysis, infect two groups of mice (5 mice per group) using planktonic cells or biofilm state cells at a dose of 3 × 107 colony-forming units (CFU), and another 5 mice were injected with PBS as a control.
    2. For detection of mRNA expression of TLR2 and cytokines in brain, infect two additional groups of mice (6 mice per group) using planktonic cells or biofilm state cells at a dose of 3 × 107 CFU.
    3. Determine the number of viable bacteria by plating serial dilutions on to THA.
    4. Euthanize all mice at 12 h post-infection for further research.
      ​NOTE: Glycerol in the stock medium must be removed before infection. Both planktonic cells and biofilm state cells recovered from -80 °C are washed 3 times with PBS. Then, planktonic cells are resuspended with PBS and diluted to the appropriate dose for infection; biofilm state cells are resuspended with the stored supernatant (from step 1.2.5) and diluted to the appropriate dose for infection. The volume for infection should not be more than 50 µL.
  2. Detecting the mRNA expression of TLR2 and cytokines in brain tissue
    1. Extract the total RNA from brain tissue using a RNA extraction kit.
      1. For each mouse, use whole brain tissue for extracting RNA. Divide whole brain tissue into 5 tubes. Take up to 100 mg brain tissue and add 1 mL of lysis solution to each tube containing the lysing matrix provided by the kit.
      2. Process the tube in a homogenizer for 40 s at a setting of 6.0, and then centrifuge the tube at 12000 × g and 4 °C for 5 min.
      3. After centrifugation, transfer the upper phase to a new microcentrifuge tube.
      4. Incubate the transferred sample for 5 min at room temperature; add 300 µL of chloroform, vortex for 10 s, and then incubate for 5 min at room temperature.
      5. Centrifuge the tube at 12000 × g and 4 °C for 5 min. Transfer the upper phase to a new tube.
      6. Add 500 µL of cold absolute ethanol to the tube, before inverting it for 5 times, and then store the sample at -20 °C for at least 1 h.
      7. Centrifuge the tube at 12000 × g and 4 °C for 15 min and remove the supernatant. Wash the pellet with 500 µL of cold 75% ethanol in RNase-free H2O.
      8. Remove the ethanol, air dry the pellet for 5 min at room temperature, and resuspend the RNA in 100 µL of RNase-free H2O.
      9. Incubate the RNA for 5 min at room temperature and determine the RNA concentration using a RNA bioanalyzer.
    2. Perform cDNA synthesis, including elimination of genomic DNA (gDNA), using a thermocycler with a reverse transcription (RT) reagent kit.
      1. Add 1 µg of RNA to a tube containing 2 µL of 5x gDNA elimination buffer, 1 µL of gDNA elimination enzyme and RNase-free H2O until the volume reaches 10 µL. Incubate for 2 min at 42 °C.
      2. Add 10 µL of master mix (1 µL of RT enzyme Mix I, 1 µL of RT Primer Mix, 4 µL of 5x Buffer 2, and 4 µL of RNase-free H2O) to the reaction solution from the last step and then mix gently. Proceed immediately with the RT reaction: 37 °C for 15 min and then 85 °C for 5 s.
      3. Perform the quantitative real-time PCR (RT-qPCR) analysis using a Real-Time PCR machine with a SYBR RT-qPCR kit. Add 10 µL of 2x enzyme, 0.8 µL of forward primer, 0.8 µL of reverse primer, 0.4 µL of 50x ROX Reference Dye II, 2 µL of cDNA template, and RNase-free H2O until a total volume of 20 µL.
      4. Run each sample in triplicate using the following thermal parameters: 30 s at 94 °C, followed by 40 cycles of 5 s at 95 °C, and 34 s at 60 °C, with a final stage of 15 s at 95 °C, 1 min at 60 °C, and 15 s at 95 °C. Primers for RT-qPCR are listed in Table 1. Housekeeping genes B2m and 18s rRNA were used as the internal controls.
    3. Calculate the relative fold change based on the 2-ΔΔCt method.
  3. Observation of histological sections
    1. After 24 h fixation with 10% formalin, reconstruct the fixed brain tissue into appropriate sizes of tissue pieces at 2 - 3 mm.
    2. Put the tissue pieces in an embedding cassette and immerse in a new fixative solution for dehydration with an automatic vacuum dehydrator.
    3. Take out the fixed tissue and dehydrate it sequentially in increasing concentrations of ethanol (70%, 80%, 95%, 95%, and 100%) for 30 min in each solution.
    4. Transparentize the sample with xylene for 15 min, dip it in liquid paraffin at 60 °C for 90 min, and then embed using an embedding machine.
    5. Cut the paraffin block embedded tissue into 3 µm slices, tile the slice on water at 45 °C, and then dry it in the air.
    6. Incubate the section for 3 h at 60 °C, stain it using the haematoxylin and eosin (HE) method, and seal it with the neutral gum.
    7. Observe the histopathological changes of the brain with an optical microscope. A four-point grading system was used to evaluate histological changes. Use an unpaired t test for statistical analysis.
      Congestion/haemorrhage: absent, score 0; small area of focal congestion/haemorrhage, score 1; large area of focal congestion/haemorrhage, or multiple sites of small area of focal congestion/haemorrhage, score 2; up to three large focal congestion/haemorrhage sites, score 3; diffuse congestion/haemorrhage, score 4.
      Necrosis: absent, score 0; focal necrosis, score 1; diffuse occurrence of 1 - 10 necrotic cells, score 2; foci of confluent necrosis, score 3; extensive confluent necrosis, score 4.
      Inflammatory cell infiltration: absent, score 0; few and focal infiltrations, score 1; multifocal infiltration or infiltrations with formation of aggregates, score 2; up to three aggregates, score 3; and more than four aggregates, score 4.

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Results

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,...

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Discussion

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 ...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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].

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Todd Hewitt Broth(THB)Becton, Dickinson and CompanyDF0492078Dissolve 30 g of the powder in 1 L of purified water. Autoclave at 121° for 15 min.
AgarDSBIO16C0050Dissolve 15 g of the powder in 1 L of THB. Autoclave at 121° for 15 min.
Milli-Q Reference Water Purification SystemMerck KGaAZ00QSVCUSWithout Dnase/ Rnase
NaClTianjin Kemiou Chemical Reagent Co., Ltd10019318Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121° for 15 min. Use KOH to adjust pH to 7.4.
Na2HPO3Xilong Scientific Co., Ltd9009012-01-09Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121° for 15 min. Use KOH to adjust pH to 7.4.
KClXilong Scientific Co., Ltd9009017-01-09Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121° for 15 min. Use KOH to adjust pH to 7.4.
KH2PO4Xilong Scientific Co., Ltd9009019-01-09Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121° for 15 min. Use KOH to adjust pH to 7.4.
KOHXilong Scientific Co., Ltd9009014-01-09Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121° for 15 min. Use KOH to adjust pH to 7.4.
GlycerolSionpharm Chemical Reagent Co., Ltd10010618Diluted with equal volumu of purified water, autoclave at 121° for 15 min
4% paraformaldehydeSionpharm Chemical Reagent Co., Ltd80096675
25% GlutaraldehydeSionpharm Chemical Reagent Co., Ltd3009243610-fold diluted with purified water for fixation.
EthanolSionpharm Chemical Reagent Co., Ltd10009218
ChloroformSionpharm Chemical Reagent Co., Ltd10006818
SpctrophotometreDeNovix Inc.DS-11+
Ultrasound cell crusherNingBo Scientz Biotechnology Co.,LtdJY96-IIN
CentrifugeHitachi Koki Co., LtdCT15RE
RefrigeratorAucma Co., LtdDW-86L500
Scanning electron microscopeZeissEVO-LS10
FastRNA Pro Green KitMP Biomedicals#6045-050
FastPrep-24 InstrumentMP Biomedicals116005500
Instrument for PCRSensoQuest GmbH1124310110
QuantStudio 6 FlexThermo Fisher Scientific4485689
SYBR Premix Ex Taq IITakara Biomedical Technology (Beijing) Co., LtdRR820A
PrimeScript RT reagent kit with gDNA EraserTakara Biomedical Technology (Beijing) Co., LtdRR047A
Fully Enclosed Tissue ProcessorLeica Biosystems Nussloch GmbHASP200S
Heated Paraffin Embedding ModuleLeica Biosystems Nussloch GmbHEG1150H
Semi-Automated Rotary MicrotomeLeica Biosystems Nussloch GmbHRM2245
Water bath for paraffin sectionsLeica Biosystems Nussloch GmbHHI1210
Autostainer XLLeica Biosystems Nussloch GmbHST5010
Agilent 2100Agilent TechnologiesG2939A
Optical microscopeOlympusBX51

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Biofilm MeningitisPlanktonic CellsBiofilm State CellsHistopathological AnalysisTLR2 ExpressionCytokine AnalysisRNA Extraction
Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model
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