グループBのマウスモデル<em&gt;連鎖球菌</em&gt;膣植民地化

要約

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

要約

成人の30％ - ストレプトコッカス・アガラクティエ （B群連鎖球菌 、GBS）は、消化器および10の膣管のグラム陽性、無症候性植民です。新生児、妊婦、高齢者を含む免疫無防備状態の個体では、GBSは、敗血症、関節炎、肺炎、および髄膜炎を引き起こす侵襲性の病原体に切り替えることができます。 GBSは、新生児の主要な細菌性病原体であるため、現在の予防は、GBS膣コロニー形成およびGBS陽性の母親のその後の周産期抗生物質による治療のための妊娠後期のスクリーニングから構成されています。ヘビーGBS膣の負担が新生児疾患と植民地化の両方の危険因子です。残念ながら、少しはホストと促進またはGBS膣コロニー形成を可能にする細菌の要因について知られています。このプロトコルは、細菌ロアを決定するために、単一のβエストラジオール前処置と毎日のサンプリングを使用して永続的なGBS膣コロニー形成を確立するための技術を説明しますD。また、さらに詳細な方法関心の追加の治療薬または試薬を投与すると、膣洗浄液および生殖管の組織を収集します。このマウスモデルは、妊娠中の母体の膣コロニー形成を制御し、脆弱な新生児への送信を防止するために、潜在的な治療標的につながる膣環境内GBS-ホスト相互作用の理解を促進します。また、女性の膣管内の一般的な細菌宿主相互作用の我々の理解を高めるために対象としています。

概要

ストレプトコッカス・アガラクティエ 、B群連鎖球菌 （GBS）は、頻繁に腸と健康な成人の尿生殖路から隔離されているカプセル化された、グラム陽性細菌です。 1970年代には、GBSは毎年、新生児の病気の7,000以上の例¹で、感染新生児死亡率の主要な薬剤として登場しました。早期発症GBS疾患（EOD）が肺炎や呼吸困難として生じる、最初の数時間または生命の日に発生し、多くの場合、遅発性疾患（LOD）のに対し、敗血症に発展する数ヶ月後に続いて起こると頻繁に菌血症、と提示します²髄膜炎に進みます。 2002年のように、疾病管理予防センター（CDC）は、GBS陽性の母親¹に妊娠後期や分娩に抗生物質予防法（IAP）でGBS膣植民地化のための普遍的なスクリーニングを推奨しています。毎年IAPによる米国では約千例に早期発症疾患の減少にもかかわらず、GBSは、早発型新生児敗血症の主要な原因のままであり、遅発性の発生は¹影響を受けないままです。子宮内かどうかは、労働者の間に、あるいは遅発性の場合には、GBSの新生児の暴露は髄膜炎、高度に規制血液の交差した場合に、ホスト環境や障壁、免疫回避の数を介して生存、横断を必要とし、脳関門^2。新生児内のこれらの病原性の相互作用の上流には、母体の膣管の初期の植民地です。母体のGBS膣コロニー形成率は^12.7％3,4の推定平均速度で、先進国と途上国での8から18パーセントの範囲です。妊娠中の膣管のGBSコロニー形成は、個々の女性の⁵の間で自然の中で、一定の断続的な、または一過性であってもよいです。興味深いことに、母体の年齢> 36年には、永続的植民地化⁶に関連しています。 GBS膣植民地化のための多数の生物学的および社会経済的なリスク要因同定されています。生物学的因子は、胃腸GBSのコロニー形成および腸内で乳酸桿菌の不在が含まれます。しかし、民族性、肥満、衛生、および性的活動もGBS膣キャリッジ⁷に関連しています。

新生児の感染を引き起こすための悪名高いが、GBSはまた、母体の感染症の両方の周産期および産後の様々な原因となります。 GBSキャリッジも疾患実体⁹とすることができる、いくつかのケースでは、膣炎⁸を呈する女性で増加しています。また、妊娠中の生殖器官のGBSの昇天は、羊膜内感染症または絨毛羊膜炎¹⁰になることがあります。また、妊娠の3.5％までに、GBSは、尿路感染症や無症候性細菌尿^11を引き起こすために膀胱に発信しています。妊娠中のGBS細菌は、分娩の発熱、絨毛羊膜炎、早産、およびprematurのリスク増加と関連しています膜¹²の電子破裂。まとめると、膣管内のGBSの存在は、複数の宿主組織の感染症にリンクされており、このニッチからGBSを排除する能力の両方の妊産婦と新生児の健康のために不可欠です。

最近まで、頸膣管とGBSの相互作用を調べる作業の大半は、in vitro細胞モデル^13-15に制限されていました。これらのin vitroの実験では、表面タンパク質を含む接着のために重要である細菌の要因を、明らかにしたような線毛およびセリンリッチリピート^17,18と同様に、二成分調節系^15,19と膣上皮のグローバルな転写応答GBS ^19。しかし、完全に膣路内にホスト微生物の相互作用を解明するために、堅牢な動物モデルが必要です。初期の研究は、GBSは、接種したマウス^20,21およびラットの膣管から回収することができることを実証しました^{両方の妊娠と非妊娠状態の22。 2005年には、短期的なGBS膣植民地化は、24時間の期間23にわたって膣GBSを治療するためのファージ溶菌酵素の有効性を調べるために、マウスでモデル化しました。数年後、長期GBS膣植民地化のマウスモデルは、GBSの永続性を管理するホストおよび細菌の要因を研究するために開発されました。このモデルは、表面付属17,18およびGBS 2成分系19,24を含むコロニー形成に貢献する数々のGBS因子を同定しました。このモデルは、ホスト応答機構19,25の同定に貢献してきたし、免疫調節ペプチド26とプロバイオティクス27を含むいくつかの治療戦略をテストするために使用されました。このプロトコルは、マウスの膣管内にGBSを接種し、その後コロニー形成を追跡し、さらなる分析のためのサンプルを収集するために必要な指導を提供します。}

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プロトコル

All animal work was approved by the Office of Lab Animal Care at San Diego State University and conducted under accepted veterinary standards. Female mice, age 8 - 16 weeks, were used for the development of this method.

1. Preparation and Intraperitoneal Injection of β-estradiol

1. Measure out β-estradiol (0.5 mg/mouse) on weigh paper while wearing appropriate personal protective equipment (PPE). CAUTION: β-estradiol can be absorbed through the skin and mucosal surfaces.
2. Transfer β-estradiol to a 15-ml conical tube and vortex until all lumps are removed and the β-estradiol is a fine powder. Draw up sesame oil (100 µl/mouse) into a 10-ml syringe. Syringe-filter sesame oil into the 15-ml conical tube containing the β-estradiol using a 0.45-µm filter. Vortex the 15-ml conical tube until the β-estradiol is a homogenous suspension in the sesame oil.
3. Draw up the β-estradiol suspension into a new 10-ml syringe. With an 18 G, 1-in. needle, aliquot 100 µl of the suspension into 1-ml tuberculin syringes. Prepare one syringe for each mouse. Place a new, sterile 26 G, ½-in. needle on each tuberculin syringe.
4. Administer 0.5 mg of the β-estradiol suspended in 100 µl of sesame oil (5 mg/ml) to each mouse 24 hr prior to bacterial inoculation. Inject each mouse within the peritoneal cavity, in the lower abdominal quadrants, just to the right or left of the midline, as previously described²⁸.
   NOTE: No cleaning or clipping of the injection site is necessary.

2. Vaginal Inoculation with GBS

1. One day prior to inoculation, grow a 5-ml overnight liquid culture of a GBS strain of interest, such as A909 (serotype Ia), in Todd Hewitt broth (THB) at 37 °C.
2. Subculture the GBS overnight culture at a 1:10 volume into fresh THB and incubate at 37 °C. Grow the bacteria to mid-log phase (OD₆₀₀ = 0.4-0.5). NOTE: This will typically take 2 - 3 hr, depending on the strain of GBS.
3. Transfer the subculture to a sterile 15-ml conical tube and pellet bacteria at 3,000 × g for 5 min. Aspirate the supernatant. Resuspend the bacterial pellet in 200 µl of sterile phosphate-buffered saline (PBS).
4. Using the resuspended pellet, bring 3 - 5 ml of PBS (1 ml per 10 mice) to exactly OD₆₀₀ = 0.4 in a new 5-ml culture tube. This will be a concentration of ~ 1 × 10⁸ colony forming units (CFU)/ml. Transfer to a new 15-ml conical tube and re-pellet the bacteria at 3,000 × g for 5 min. Aspirate the supernatant.
5. Resuspend the pellet in PBS at 1/10 the original volume. For example, if 3 ml of OD₆₀₀ = 0.4 was pelleted, then resuspend it in 300 µl of PBS. NOTE: This is the final bacterial suspension (~ 1 × 10⁹ CFU/ml) used for animal inoculation.
6. Reserve 50 µl of this suspension for serial dilution and plating on THB agar to determine the exact inoculum.
7. Inoculate each mouse with 10 µl of the final bacterial suspension so that 1 × 10⁷ CFU is administered to each mouse.
   1. To inoculate, restrain the mouse manually by securing the loose skin at the scruff of the neck between the handler's thumb and index finger and then immobilizing the tail, as described previously²⁸.
   2. Draw up 10 µl of the GBS prepared in step 2.6 into a 200-µl gel loading pipette tip. Insert the tip 5 to 10 mm into the vaginal lumen and dispense the 10 µl of inoculum.
      NOTE: Gel loading tips are preferred over standard 200-µl tips to minimize the risk of organ trauma or injury, particularly in younger or smaller mice.
   3. Immediately following inoculation, release the scruff of the neck and elevate the hind end of the mouse, lifting the mouse by the tail and walking the front paws on a hard surface for ~ 1 min.
   4. Visually inspect the vaginal opening for any backflow of inoculum. If backflow is observed, a fresh pipette tip may be used to manipulate or enlarge the vaginal opening, facilitating uptake of the backflow into the lumen. Additionally, backflow may be aspirated via pipette and re-inoculated.
      NOTE: If administering topical agents, probiotic organisms, or proteins of interest, a volume up to 20 µl in a physiologic buffer may be given in the vaginal tract.

3. Swabbing the Vaginal Lumen to Quantify GBS Load

1. Prepare one 1.5-ml micro-centrifuge tube per mouse by adding 100 µl of PBS. Just prior to swabbing, pre-wet the swab in PBS.
2. Restrain the mouse as described in step 2.7.1 and insert the swab 10 mm into the vaginal lumen. Gently rotate the swab 4 times clockwise and 4 times counter clockwise, applying slight pressure to the vaginal wall.
3. Transfer the swab to the 1.5-ml microcentrifuge tube with 100 µl of PBS. Prior to plating, vortex the microcentrifuge tube for ~15 sec to release the bacteria from the swab. Serially dilute each sample in PBS and plate 20 µl of dilutions 1:10 through 1:10,000 on differential medium agar plates prepared per the manufacturer's instructions. Incubate the plates at 37 °C for 24 hr. GBS colonies will appear either bright pink or mauve in color. Other endogenous flora will be inhibited, or will appear as blue, white, or grey colonies.

4. Collecting Vaginal Lavage Fluid

1. Restrain the mouse as described in step 2.7.1. Using a 200-µl gel loading pipette tip, pipet 20 µl of PBS into the vaginal lumen. Gently pipet the entire volume up and down 4 times within the lumen, and then withdraw the entire volume in the same pipette tip. NOTE: If the lavage fluid is thick with mucous, a standard 200 µl pipette tip can be used to collect the final lavage fluid.
2. If saving lavage fluid for cytokine analysis or CFU quantification, dispense into a 0.7-ml microcentrifuge tube. If determining the stage of estrus, dispense at least 5 µl of lavage fluid onto a glass slide and observe the cells under 100X magnification on a light microscope. NOTE: For examples of estrous stages, see Figure 1.

5. Tissue Dissection and Homogenization

1. For each mouse, prepare three 2-ml screw cap tubes (one for each tissue: vagina, cervix, and uterus). Fill each tube with ample (0.4 - 0.5 g) 1.0 mm zirconia beads to cover the conical-shaped bottom of the tube.
2. Autoclave the prepared tubes prior to collecting tissue for 30 min at 121 °C, especially if quantifying bacterial load. Add 500 µl of sterile PBS to each tube. Weigh each tube after autoclaving and record for future reference to calculate recovered tissue weight.
3. Sacrifice the mouse using approved institutional methods such as CO₂ asphyxiation and cervical dislocation. Spray down the ventral abdomen with 70% EtOH. With sterile scissors, open the abdominopelvic cavity, lift the back skin and abdominal muscles, and displace the intestines so that the reproductive tract is exposed.
4. Sterilize the scissors with 70% EtOH, wipe clean if necessary, and cut both uterine horns mid-length between the uterine body and ovaries. Using scissors and forceps, separate visceral fat, membranes, and the urinary bladder away from the reproductive tract, moving caudally.
5. Sterilize scissors as described in step 5.4. Transversely cut the vagina as close to the vulva as possible to separate the reproductive tract from the body. Lift and remove the intact reproductive tract and place it in a sterile Petri dish.
6. Using a new razor blade, separate the uterus from the cervix in one transverse cut. Sterilize the razor blade with 70% EtOH, and then separate the cervix from the vagina in one transverse cut. NOTE: There will be a minimal amount of uterine tissue still attached to the cervix. There will be a minimal amount of vaginal tissue still attached to the cervix.
7. Using sterile forceps, transfer each of the tissues into their respective 2-ml tubes containing PBS and homogenizing beads. Clean the forceps with 70% EtOH in between handling each tissue.
8. Weigh each 2-ml screw cap tube and subtract the original weight of tube to determine the tissue weight. Tissue weights typically vary between 20 - 100 mg. Tightly seal the screw cap tubes and homogenize the tissues for 1 min at maximum speed in a tissue homogenizer.
9. To quantify bacterial load, serially dilute 25 µl of tissue homogenate and plate dilutions 1:10 through 1:10,000 on differential medium agar plates. Incubate the plates as described in step 3.3. To store samples for cytokine quantification, freeze tissue homogenates at -20 °C.

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結果

During the development of this model, multiple observations were made regarding factors that affect the duration of GBS vaginal colonization. To determine how estrous stage at inoculation impacts GBS bacterial persistence, mice were staged on the day of inoculation via vaginal lavage fluid. Figure 1 illustrates the four stages of the mouse estrous cycle, as determined by wet-mount vaginal lavage fluid, a well-established method²⁹. Mice were divided into groups ...

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ディスカッション

To further the advancement of the understanding of GBS interactions with the both the host and other microbes within the context of the host, an animal model is required. This work describes the technical aspects of establishing GBS vaginal colonization in mice. This protocol achieves > 90% colonization of mice without the use of anesthetics to inoculate bacteria or to collect swab samples, immune-suppressants to enable colonization, vaginal pre-washing, or additives to thicken the inoculum. Moreover, this model demo...

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資料

Name	Company	Catalog Number	Comments
Sesame oil	Sigma Aldrich	S3547-250ML
β-Estradiol	Sigma Aldrich	E8875-1G	CAUTION: Wear appropriate PPE. β-estradiol can be absorbed through the skin and mucosal surfaces.
200 μl gel loading pipette tips	USA Scientific	1252-0610
Urethro-genital, sterile, calcium alginate swabs	Puritan	25-801 A 50
CHROMagar StrepB	DRG International	SB282
Todd Hewitt Broth	Hardy Diagnostics	7161C
18 G, 1.5 inch needles	BD	305199
26 G, 0.5 inch needles	BD	305111
10 ml syringes	BD	309604
1 ml syringes	BD	309659
0.45 μm PVDF syringe filters	Whatman	6900-2504
Dulbecco's Phosphate-Buffered Salt Solution 1x	Corning	21-031-CV