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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single β-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

Introduction

Streptococcus agalactiae, group B Streptococcus (GBS), is an encapsulated, Gram-positive bacterium which is frequently isolated from the gut and genitourinary tract of healthy adults. In the 1970s, GBS emerged as the leading agent of infectious neonatal mortality, with over 7,000 cases of neonatal disease annually1. Early-onset GBS disease (EOD) occurs in the first hours or days of life, arises as pneumonia or respiratory distress, and often develops into sepsis, whereas late-onset disease (LOD) ensues after several months and presents with bacteremia, which frequently advances to meningitis2. As of 2002, the Centers for Disease Control and Prevention recommends universal screening for GBS vaginal colonization in late gestation and intrapartum antibiotic prophylaxis (IAP) to GBS-positive mothers1. Despite the reduction of early-onset disease to approximately 1,000 cases in the United States annually due to IAP, GBS remains the leading cause of early-onset neonatal sepsis, and late-onset occurrence remains unaffected1. Whether in utero, during labor, or even in late-onset cases, neonatal exposure to GBS requires survival, transversal through a number of host environments and barriers, immune evasion, and, in the case of meningitis, crossing of the highly regulated blood-brain barrier2. Upstream of these virulent interactions within the neonate is the initial colonization of the maternal vaginal tract. Maternal GBS vaginal colonization rates range from 8-18% in developed and developing countries, with an estimated average rate of 12.7%3,4. GBS colonization of the vaginal tract during pregnancy may be constant, intermittent, or transient in nature among individual women5. Interestingly, a maternal age > 36 years is associated with persistent colonization6. Numerous biological and socio-economical risk factors for GBS vaginal colonization have been identified. Biological factors include gastrointestinal GBS colonization and absence of Lactobacillus within the gut. However, ethnicity, obesity, hygiene, and sexual activity have also been associated with GBS vaginal carriage7.

Although notorious for causing neonatal infections, GBS also causes a variety of maternal infections both peripartum and postpartum. GBS carriage is increased in women presenting with vaginitis8 and, in some cases, may even be the disease entity9. Additionally, GBS ascension of the reproductive tract during pregnancy may result in intra-amniotic infection or chorioamnionitis10. Moreover, in up to 3.5% of pregnancies, GBS disseminates to the urinary bladder to cause a urinary tract infection or asymptomatic bacteriuria11. GBS bacteriuria during pregnancy is associated with an increased risk of intrapartum fever, chorioamnionitis, preterm delivery, and premature rupture of membranes12. Taken together, the presence of GBS within the vaginal tract is linked to infections of multiple host tissues, and the ability to eliminate GBS from this niche is imperative for both maternal and neonatal health.

Until recently, the majority of work examining GBS interactions with the cervicovaginal tract was limited to in vitro cell models13-15. These in vitro experiments have revealed bacterial factors that are important for adherence, including surface proteins such a pili and serine-rich repeats17,18, as well as two-component regulatory systems15,19 and the global transcriptional response of the vaginal epithelium to GBS19. However, to fully elucidate the host-microbe interactions within the vaginal tract, a robust animal model is necessary. Early work demonstrated that GBS can be recovered from the vaginal tract of inoculated mice20,21 and rats22 in both pregnant and non-pregnant conditions. In 2005, short-term GBS vaginal colonization was modelled in mice to examine the efficacy of a phage lytic enzyme to treat vaginal GBS over a 24 hr period23. Several years later, a long-term GBS vaginal colonization mouse model was developed to study host and bacterial factors governing GBS persistence. This model has identified numerous GBS factors contributing to colonization, including surface appendages17,18 and GBS two-component systems19,24. This model has contributed to the identification of host response mechanisms19,25 and was used to test several therapeutic strategies, including immunomodulatory peptides26 and probiotics27. This protocol gives the necessary guidance to inoculate GBS into the mouse vaginal tract and to subsequently track colonization and collect samples for further analyses.

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Protocol

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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Results

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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Discussion

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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Disclosures

The authors have nothing to disclose.

Acknowledgements

We would like to thank the vivarium manager and staff at San Diego State University for support with animal husbandry. During this work, K.A.P. was supported by an ARCS scholarship and a fellowship from the Inamori Foundation. K.S.D. is supported by an R01 grant, NS051247, from the National Institutes of Health.

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Materials

NameCompanyCatalog NumberComments
Sesame oil Sigma AldrichS3547-250ML
β-Estradiol Sigma AldrichE8875-1GCAUTION: Wear appropriate PPE. β-estradiol can be absorbed through the skin and mucosal surfaces. 
200 μl gel loading pipette tips USA Scientific1252-0610
Urethro-genital, sterile, calcium alginate swabsPuritan25-801 A 50
CHROMagar StrepBDRG InternationalSB282
Todd Hewitt BrothHardy Diagnostics7161C
18 G, 1.5 inch needlesBD305199
26 G, 0.5 inch needlesBD305111
10 ml syringesBD309604
1 ml syringesBD309659
0.45 μm PVDF syringe filtersWhatman6900-2504
Dulbecco's Phosphate-Buffered Salt Solution 1xCorning21-031-CV

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