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. Author manuscript; available in PMC: 2019 Nov 26.
Published in final edited form as: Methods Mol Biol. 2019;1997:403–412. doi: 10.1007/978-1-4939-9496-0_23

A Natural Mouse Model for Neisseria Persistent Colonization

Katherine Rhodes 1, Mancheong Ma 1, Magdalene So 1
PMCID: PMC6878645  NIHMSID: NIHMS1059894  PMID: 31119636

Abstract

We have developed a natural mouse model to study persistent colonization by commensal Neisseria. The system couples the ordinary lab mouse with Neisseria musculi (Nmus), a commensal in the oral cavity and gut of the wild mouse, Mus musculus. The pairing of Nmus with its natural reservoir circumvents host restriction barriers that have impeded previous studies of Neisseria in vivo behavior. The model allows, for the first time, for the dissection of host and neisserial determinants of asymptomatic colonization. Inoculation procedures are noninvasive and susceptibility to Nmus colonization varies with host genetic background. In colonized mice, bacterial burdens are detectable up to 1-year post inoculation, making it an ideal model for the study of persistence. As Nmus encodes several Neisseria gonorrhoeae (and Neisseria meningitidis) host interaction factors, the system can be used to query the in vivo functions of these commonly held genes and factors. Nmus also encodes many pathogenic Neisseria vaccine targets including a polysaccharide capsule, making the model potentially useful for vaccine development. The ease of genetic manipulation of Nmus enhances the feasibility of such studies.

Keywords: Mouse model, Colonization and persistence, Commensal Neisseria, Pathogenic Neisseria

1. Introduction

1.1. Why Is It Important to Understand Commensalism?

Commensal organisms (microbiota, normal flora) play a critical role in physiology [1–7]. They are required for gut and immune system development, and prevent pathogen colonization. Perturbation of the composition of these microbial communities is strongly linked to chronic, non-infectious diseases such as obesity, inflammatory bowel disease, diabetes (Type I and II) and autoimmunity.

How does a commensal organism colonize and persist in the body in the presence of a robust immune system? Little is known about the mechanisms of commensal–host coadaptation. Understanding the molecular basis of these interactions will improve disease prevention and contribute to our fundamental knowledge of immunology and microbiology. Except for two species, all members of the Neisseria genus are commensals that inhabit a wide range of hosts, from rodents, felines, and bovines to monkeys and man [8, 9]. Animal-adapted commensal Neisseria have been detected or isolated from the oral cavity and feces of their hosts, suggesting they establish niches along the alimentary tract. The niche of human-adapted commensals is generally assumed to be the upper respiratory tract, but little has been done to examine the extent of their colonization range. The two pathogenic species in the genus are Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis (Nme), which only infect humans [8]. Ngo and Nme inhabit the oral cavity and urogenital tract and are generally assumed to be restricted to these sites. Whether they also colonize the gut is an open question.

Ngo and Nme exhibit a commensal-like behavior; at fairly high frequency, they colonize the oral cavity and urogenital tract without eliciting overt symptoms of disease [10, 11] (http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.177.8777). Asymptomatic carriage is an important facet of infection, as it is key to person-to-person transmission. The mechanisms that promote asymptomatic carriage of Ngo and Nme are little understood. As commensal Neisseria are closely related to Ngo and Nme, they may provide a means to understanding asymptomatic infections by these pathogens.

1.2. A Natural Mouse Model for Commensal Neisseria Persistent Colonization

We have developed a natural small animal model for dissecting the mechanisms of Neisseria colonization and persistence, from the standpoint of both host and microbe [12]. Our model pairs the lab mouse, which does not normally harbor Neisseria, with Neisseria musculi (Nmus) a commensal of wild mice [13]. Pairing the mouse with a member of its normal flora circumvents barriers imposed by host restriction, a problem that plagues current models for studying the human-specific pathogenic Neisseria species [14–17] (see Note 1).

Nmus encodes many host interaction genes of the pathogenic Neisseria, including a complete set of genes for Type IV pilus (Tfp) biogenesis and function [12]. The Tfp fiber is generally assumed to mediate colonization [18, 19], based on in vitro studies, but in vivo function has never been tested. As final proof of principle for the validity of our model for investigating Neisseria-host interactions, the role of Tfp in colonization was tested using the mouse model. Nmus ΔpilE, which cannot produce the Tfp fiber subunit, PilE, fails to colonize CAST and B6 mice [12]. The Nmus-mouse model is therefore useful for querying the in-host function of other proteins that modulate Tfp activity, and the interaction genes/factors held in common by commensal and pathogenic Neisseria (see Note 2).

Finally, it should be noted that commensal Neisseria naturally colonize rhesus macaques (RM) [20] and persist in them for at least 6 years [21]. RM Neisseria are closely related to human commensal species [22] and encode many host interaction factors of the pathogenic species [21] (Weyand and So unpublished). Thus, pairing RM Neisseria with the macaque holds promise for understanding Neisseria-host interactions. However, the logistics and expense of the model, and the limited number of non-human primate facilities make the model inaccessible to most investigators. In this chapter, we provide a detailed protocol for Nmus colonization of mice that is readily accessible to investigators.

2. Materials

2.1. Screen for Autochthonous Neisseria Species

2.1.1. Sample Collection

  1. Mouse (see Note 3).

  2. Ear tag system or ear punch.

  3. 100 μl aliquots of sterile Dulbecco’s phosphate buffered saline without magnesium and calcium (DPBS).

  4. 1.5 ml Sterile Eppendorf tubes.

  5. BBL CultureSwab Plus Transport system, Amies gel with charcoal.

  6. Laminar flow hood: sterilize by swabbing with 70% (v/v) ethanol in distilled H2O.

  7. Sterile catch pan.

2.1.2. Oral Sample Preparation

  1. Gonococcal Medium Base (GCB) agar plate (BD Difco): add 15 g/L of Proteose Peptone No. 3, 1 g/L of Cornstarch, 4 g/L of K2HPO4, 1 g/L of KH2PO4, 5 g/L of NaCl, 10 g/L of agar to 1 L of distilled H2O. Autoclave at 121 °C and 1.05 kg/cm2 for 40 min. Cool the medium to 55 °C and add 1% (v/v) Kellogg’s Supplement I and 0.1% (v/v) Kellogg’s Supplement II and appropriate antibiotics (see Note 4).

  2. Kellogg’s Supplement I: 400 mg/ml of glucose, 10 mg/ml of l-glutamine, 20 μg/ml of thiamine pyrophosphate dissolved in distilled H2O and sterilized by filtration (0.22 μm filter). Store in aliquots at −20 °C.

  3. Kellogg’s Supplement II: 500 μg/ml of Iron (III) Nitrate in distilled H2O and sterilized by filtration (0.22 μm filter). Store in aliquots at −20 °C [23].

  4. Humidified incubator set at 37 °C with 5% (v/v) CO2.

  5. Sterile plating beads or glass spreader rod.

  6. GC broth (GCBL): add 15 g/L of Proteose peptone #3, 1 g/L of KH2PO4, 4 g/L of K2HPO4, 1 g/L of NaCl, and 20% (v/v) glycerol to 1 L of distilled H2O, and adjust pH to 7.2 [23]. Autoclave at 121 °C and 1.05 kg/cm2 for 40 min.

  7. Wire cutters.

  8. Plate spinner.

  9. Bunsen burner.

  10. Vortex mixer.

  11. 70% (v/v) ethanol.

  12. Glass spreader rod or disposable inoculating plastic loops.

  13. 1.5 ml sterile Eppendorf tubes.

2.1.3. Fecal Sample Preparation

  1. Same materials as described in Subheading 2.1.2.

  2. Sterile P1000 pipette tips.

  3. Tared balance.

2.2. Preparing the Neisseria musculi Inoculum

  1. Neisseria musculi strains/isolates, stored at −80 °C in GCBL with 20% (v/v) glycerol.

  2. GCB agar plates containing 40 μg/ml of rifampicin, and any additional selective antibiotics appropriate to the strain being tested.

  3. Sterile CLASSIQSwabs (Copan, or equivalent).

  4. Sterile plastic cell scrapers.

  5. DPBS without calcium and magnesium.

  6. GCBL broth.

  7. Humidified incubator set at 37 °C with 5% (v/v) CO2.

  8. Dissecting microscope.

  9. Spectrophotometer.

  10. Disposable plastic cuvettes.

  11. 1.5 ml sterile Eppendorf tubes.

  12. Vortex mixer.

2.3. Inoculation of Neisseria musculi into the Oral Cavity

  1. Prescreened mice with Identification (ID) numbers.

  2. Neisseria musculi inoculum.

  3. P200 pipet.

  4. Aerosol-resistant tips.

  5. Vortex mixer.

  6. Sterile catch pans.

3. Methods

3.1. Screen for Autochthonous Neisseria Species (See Note 5)

3.1.1. Sample Collection

  1. On day 1, tag or band each mouse to give it an identification (ID) number.

  2. Sterilize the laminar flow hood. Collect the necessary equipment and place it in the hood, along with a sterile catch pan and the cage of naive mice.

  3. Gently restrain the mouse so as to prevent the forelimbs from touching the mouth or nose.

  4. Submerge a BBL culture swab into an aliquot of DPBS; remove excess liquid by gently pressing the swab along the side of the tube. Collect an oral sample by gently inserting the wetted swab into the oral cavity of the mouse, taking care that it does not reach the esophagus. Using a swirling motion, pass the swab over the palate, cheek pouches, sublingual tissue, and tongue. Return the swab to the receptacle, making sure it does not touch the side of the tube. Record the mouse ID on the side of the transport tube.

  5. Use a sterile, labeled Eppendorf tube to collect a fecal sample from each mouse (see Note 6). Use the inside edge of the tube to scoop the sample into the tube, taking care not to allow the fecal pellet to contact any unsterilized surface or gloved fingers.

  6. Proceed to sample preparation and plating as described in Subheadings 3.1.2 and 3.1.3.

3.1.2. Oral Sample Preparation

  1. Dispense 1 ml of GCBL containing 20% (v/v) glycerol into prelabeled sterile Eppendorf tubes.

  2. Dip the metal cutting edge of a pair of wire cutters in 70% (v/v) ethanol and flame to sterilize. Wait for the pliers to cool.

  3. Remove the oral swab from the media receptacle in step 4 of Subheading 3.1.1 without touching the metal shaft.

  4. Insert the cotton bud of the swab into the appropriate tube with GCBL containing 20% (v/v) glycerol. Use the sterile wire cutters to clip the cotton bud from the shaft, dropping the bud into the storage media. Cap the tube firmly and vortex at high speed for 1 min.

  5. Repeat step 2 between each swab.

  6. Pulse each sample on the vortex mixer for 2 s to suspend any settled material.

  7. Dispense 100 μl of each sample onto a labeled, prewarmed GCB plate with appropriate antibiotics (see Note 4).

  8. Spread the liquid evenly over the agar surface using a sterilized glass rod or disposable loop. Incubate the plate for 48 h at 37 °C in an atmosphere with 5% (v/v) CO2.

  9. Check plates for growth of bacteria. When screening for endogenous Neisseria, exclude any mouse that is positive for either oral or fecal CFUs (see Note 7).

3.1.3. Fecal Sample Preparation

  1. Weigh each fecal sample collected from step 5 in Subheading 3.1.1 in its tube on a tared balance scale and record the weight.

  2. Dispense 1 ml of GCBL containing 20% (v/v) glycerol into each tube, taking care to prevent cross contamination.

  3. Crush each fecal pellet using a sterile filtered P1000 pipette tip. Vortex each sample for ~1 min at high speed until homogenous.

  4. Repeat steps 6–9 of Subheading 3.1.2.

3.2. Preparing the Neisseria musculi Inoculum (See Note 8)

  1. Use a sterile swab to inoculate a GCB plate containing 40 μg/ml of rifampicin with Nmus from a pure freezer stock, without allowing the stock to thaw, and streak for single colony isolation (see Note 9).

  2. Incubate the plate at 37 °C with an atmosphere of 5% (v/v) CO2 for 48 h to obtain colonies large enough to visualize colony morphological type.

  3. Use a dissecting microscope to view and collect ten rough colonies onto a single sterile swab. Streak a lawn onto a GCB plate containing 40 μg/ml of rifampicin with the collected colonies. Repeat five times, for a total of 50 colonies streaked to lawns on five separate plates. Repeat for each experimental strain.

  4. Incubate these plates for an additional 16–18 h at 37 °C with an atmosphere of 5% (v/v) CO2.

  5. Prepare a sterile Eppendorf tube with 1 ml of sterile DPBS.

  6. Harvest the plates that had been streaked to lawns in step 3. Collect the bacterial lawn from each plate using a sterile cell scraper, taking care not to gouge the agar. Inadvertent collection of agar with the lawn will skew absorbance readings.

  7. Deposit the collected lawns into the Eppendorf tube containing 1 ml of DPBS, to create a stock suspension.

  8. Vortex the suspension at maximum speed for at least 1 min to disperse any aggregates.

  9. Prepare a second Eppendorf tube with 900 μl of DPBS. Dilute the stock suspension 1/10 and vortex at high speed for an additional minute.

  10. Transfer the diluted bacterial suspension to a clean cuvette. Measure the optical density (OD) at λ600 nm using a spectrophotometer blanked against DPBS.

  11. Calculate the OD λ600 nm of the undiluted stock suspension. Calculate the total volume needed to dose all mice with 50 μl of inoculum per mouse, leaving a minimum of 100 μl excess for long-term storage. Using these values, calculate the volume of stock suspension necessary to create an inoculum where OD λ600 nm = 2.0.

  12. Prepare the inoculum as calculated in step 11, using a new sterile Eppendorf tube and the appropriate volumes of DPBS diluent and stock suspension. Vortex the inoculum for 1 min at high speed. Move on to Subheading 3.3 immediately. Samples remain viable in DPBS for approximately 4–6 h at room temperature.

3.3. Inoculation of Neisseria musculi into the Oral Cavity (See Note 10)

  1. Prepare the biosafety cabinet as described in step 2 of Subheading 3.1.

  2. Manually restrain each mouse as described in step 3 of Subheading 3.1, ensuring that the animal cannot move its head or forelimbs.

  3. Briefly vortex the inoculum to suspend any bacteria that may have settled to the bottom of the tube.

  4. Withdraw 50 μl of inoculum from the tube using a sterile filtered P200 pipette. Insert the pipette tip into the oral cavity of the restrained mouse, taking care not to abrade the tissues. Slowly dispense the inoculum. Depending on the size of the mouse, and how quickly it swallows the inoculum, this may have to be done in two stages.

  5. Continue to restrain the mouse until the majority of the inoculum has been swallowed. This will take up to 2–3 min. It is vital that the mouse does not move from an immobile, supine position before this time, to prevent inoculum loss and deviation between delivered burdens within the experimental group.

  6. Release the mouse into the sterile catch pan and repeat for the remaining mice (see Note 11).

  7. Sample mice at designated time points post inoculation following steps 3–6 of Subheading 3.1. Prepare samples for plating as indicated in Subheadings 3.1.2 and 3.1.3 or storage (see Note 4).

Acknowledgments

Funding for this project was provided by NIH 1R56A124665–01 awarded to M. So.

4. Notes

One oral dose of Nmus (6 × 106 CFUs), pipetted gently into the oral cavity of the mouse, will achieve lasting colonization of the oral cavity and gut. Nmus is recovered in large quantities from these sites every week, for as long as 1 year. The animals remain healthy throughout the studies. No administration of antibiotics or hormones, or invasive procedures or surgery is necessary. Since the system involves a commensal in its native host, mice do not need to be derived to introduce human markers, for example, receptor proteins for gonococcal virulence factors, to mimic human infection. Horizontal transmission between cage mates has not been detected, allowing the cohousing of naïve and inoculated mice.

Nmus is easy to grow and manipulate genetically; many reagents, genetic tools, and genome databases are available for both Neisseria and the lab mouse [23–25] (http://www.sanger.ac.uk/resources/download s/bacteria/neisseria.html), allowing rapid dissection of microbial and host determinants of colonization, and of the elusive process of persistence.

Host genetics is a major determinant of colonization. CAST/EiJ (CAST) and A/J strains of lab mice are colonized by Nmus at high frequency, and C57BL/6J (B6) at intermediate frequency. All studies to date have been done with mice from 4–6 weeks of age. No sex bias has been detected. NOD/LtJ, NZO/HILtJ, PWK/PhJ, WSB/EiJ, and 129S2/SvlmJ mice are resistant to colonization. A second major colonization determinant is innate immunity. B6 MyD88−/− mice are exquisitely sensitive to Nmus colonization, unlike their parental wt B6. By contrast, B6 Rag1−/− mice are no more susceptible than B6. Many genetic tools are available for manipulating the lab mouse, such as the Collaborative Cross [24], which allows for identification of mouse alleles and immune pathways that determine susceptibility/resistance to Nmus colonization.

The GCB agar formulation can be used for both prescreening and experimental sample propagation. The antibiotics used in each assay must be adapted as appropriate. To screen for autochthonous Neisseria prior to inoculation, samples must be plated on GCB agar containing 3 μg/ml of Trimethoprim and 2μ/ml of Vancomycin. This medium selects for growth of Neisseria species and discourages growth of other commensals. The experiments described in ref. 12 were done using Nmus AP2365, a spontaneously arising rifampicin-resistant strain. Samples from AP2365-inoculated mice should be selected on GCB agar containing 40 μg/ml of rifampicin, and/or the antibiotic specific to the selective marker introduced to the Nmus strain of interest. Samples can be stored at −80 °C indefinitely in GCBL with 20% (v/v) glycerol.

At least 3 days prior to inoculation, prescreen all mice for the presence of endogenous Neisseria. This is done by plating oral cavity and fecal samples on agar that selects for Neisseria species (see Note 10). This will avoid complications that interfere with interpretation of colonization data. We note that we have not detected Neisseria in any of the lab mice we have used [12]. The sampling collection and preparation method is also used for assaying experimental animals post inoculation.

If working in pairs, one person should restrain the mouse while the other collects the fecal samples and oral swabs. If working alone, the experimenter is advised to place each mouse in a sterilized container or catch basin and collect the fecal sample(s) from the animal prior to swabbing their oral cavity. This increases the efficiency of fecal sample collection, especially when working with CAST/EiJ strains.

Thorough genetic and biochemical protocols for identifying native Neisseria species can be found in ref. 13.

Preparation of the Nmus inoculum is straightforward, but good aseptic technique is crucial to prevent contamination that could confound study results. All steps should be performed in a sterile biosafety cabinet, or near a flame if working on the bench top.

Do not freeze and thaw a frozen stock of Neisseria. To avoid this problem, multiple freezer stocks should be prepared, and Nmus should be taken from frozen stock by scraping the surface of the frozen culture.

Inoculating the mouse oral cavity is a slow process; insufficient time in the oral cavity can result in artificially poor colonization efficiency and variability within experimental groups. To minimize potential variability arising from the inoculation process, all mice in the experiment should be inoculated by the same person. Mice should be inoculated and sampled at approximately the same time of day to further reduce variability between time points and experimental groups.

If inoculating multiple strains of Nmus, it is vital to sterilize the hood and instruments between groups. All materials that had contact with each group of mice should be decontaminated or replaced, before moving to the next bacterial strain to prevent cross contamination.

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