Murine Models for Staphylococcal Infection

Abstract

Staphylococcus aureus is a Gram-positive bacterium that colonizes almost every organ in humans and mice and is a leading cause of diseases worldwide. S. aureus infections can be challenging to treat due to widespread antibiotic resistance and their ability to cause tissue damage. The primary modes of transmission of S. aureus are via direct contact with a colonized or infected individual or invasive spread from a colonization niche in the same individual. S. aureus can cause a myriad of diseases, including skin and soft tissue infections (SSTIs), osteomyelitis, pneumonia, endocarditis, and sepsis. S. aureus infection is characterized by the formation of purulent lesions known as abscesses, which are rich in live and dead neutrophils, macrophages, and surrounded by a capsule containing fibrin and collagen. Different strains of S. aureus produce varying amounts of toxins that evade and/or elicit immune responses. Therefore, animal models of S. aureus infection provide a unique opportunity to understand the dynamics of organ-specific immune responses and modifications in the pathogen that could favor the establishment of the pathogen. With advances in in vivo imaging of fluorescent transgenic mice, combined with fluorescent/bioluminescent bacteria, we can use mouse models to better understand the immune response to these types of infections. By understanding the host and bacterial dynamics within various organ systems, we can develop therapeutics to eliminate these pathogens. This module describes in vivo mouse models of both local and systemic S. aureus infection.

1. Introduction

Currently, most animal models of S. aureus infection aim to 1) understand the biological role of different bacterial toxins/virulence factors from human clinical isolates, 2) provide experimental validation for the molecular basis of pathogenesis, 3) elucidate the utility of S. aureus factors as antigens for vaccines, 5) define the molecular mechanisms whereby immunity is achieved, and 6) determine the role of antimicrobials in eliminating the infection in vivo (Abdul Hamid et al., 2020; Anderson et al., 2019; Guo et al., 2013; Kim et al., 2014; Malachowa et al., 2019). More recently, an overall increased interest in understanding the role of specific immune components (innate and adaptive arms) in the pathogenesis of the disease has emerged (Brandt et al., 2018; Goldmann & Medina, 2018; Krishna & Miller, 2012). By better understanding anti-staphylococcal immune responses, we can discover targets for future therapeutic intervention to treat these infections by augmenting the host immune response.

An animal model system is a crucial tool to evaluate the efficacy of an antimicrobial agent before more extensive studies are performed on human subjects (Dejani et al., 2016; Grunenwald et al., 2018; Guo et al., 2013; Hendrix et al., 2016). To fully understand the host-microbe interaction during S. aureus infection, it is necessary to employ in vivo models that allow for consistent infection monitoring and sample collection to answer experimental questions. Here, we will be describing techniques for models of localized, invasive, and systemic S. aureus infection. These protocols provide an opportunity to study unique immune mechanisms of disease, microbial pathogenesis, vaccines, and therapeutic interventions. Basic Protocol 1 describes a method of subcutaneous skin infection. In Alternative Protocol 1, we outline a model of tape striping infection in the ear. Basic Protocol 2 outlines strategies for collecting skin tissue samples for analysis from these protocols. Basic Protocol 3 illustrates an important osteomyelitis model (an invasive infection of the bone). Finally, a systemic infection model (i.v.) is described in Basic Protocol 4. Support Protocols describe how to prepare bacterial stocks of S. aureus (Support Protocol 1) and necessary reagents/solutions (Support Protocol 2).

All experimental steps involving mice should be conducted in an animal biosafety level 2 facility, with advanced approval from an institutional animal care and use committee. Proper training of all personnel in both aseptic technique and biosafety level 2 best practices should be ensured.

Basic Protocol 1: Murine model of Staphylococcus aureus subcutaneous infection

The purpose of this protocol is to experimentally model S. aureus subcutaneous infections. SSTIs are the most common forms of S. aureus disease worldwide (Anderson et al., 2019; Malachowa et al., 2019)). As S. aureus often breaches the epidermal layer of skin, a subcutaneous model of murine S. aureus infection allows us to consistently and safely model and study these types of infections (Brandt et al., 2018). This technique allows the determination of lesion size (abscess volume and dermonecrosis) over time and sample collection (skin biopsies) of the infected area to measure bacterial burden, RNA and protein expression, histological analysis (abscess formation), and cell recruitment. Furthermore, this protocol highlights a method in which bacterial load can be monitored using in vivo animal imaging combined with fluorescent or bioluminescent bacterial strains.

Materials

• C57BL/6 mice, 6 to 10 weeks old, male or female (The Jackson Laboratory)

• Isoflurane

• O₂ Source

• 70% ethanol

• Bacterial inoculum suspended in sterile PBS (See Support Protocol 1)

• Isoflurane induction chamber with nose cone attachment

• Hair clippers

• 1-ml syringes

• ½ inch 30-gauge beveled needles

• Paper Towels

Mice preparation

• Animal clippers (ProClip clippers equipped with size 40 carbon-infused steel blade).

• Depilatory cream (Nair®).

• Forceps.

• Sterile cotton gauze pad.

• 70% Ethanol.

• Puralube® ophthalmic ointment

Preparation of the skin

• 1
  Anesthetize mouse using Isoflurane in the presence of oxygen in an induction chamber for 3–4 min, before moving the mouse to nose cone attached to Isoflurane and an O₂ source.

  1. Observe if the mice are adequately anesthetized. The animal should not respond to foot pinching.
• 2Add one drop of ophthalmic ointment in each eye.
• 3Carefully shave the mouse’s back, removing as much hair as possible using the electric clippers to enhance visualization and measurement.
• 4Gently apply Nair® for 1–3 min in the shaved area. Remove Nair® throughout the shaved area using gauze and water.
• 5Remove mice from the anesthesia and let it rest for 18–24 h in the cage.

Bacterial injection

• 6Anesthetize the previously shaved mouse in an induction chamber as described above.
• 7Remove mice from the chamber and attach the animal to the nose cone.
• 8Add one drop of ophthalmic ointment in each eye.
• 9Wipe the shaved area with 70% ethanol using a wet paper towel to establish a sterile area.
• 10
  Draw up the inoculum into the syringe, making sure to remove any bubbles from the syringe. Using Adson forceps, pinch the skin on the mouse’s back until it becomes a tent and inject 1–3 × 10⁶ S. aureus CFU or PBS (50 μl) using the 1mL syringe and a ½ inch 30-gauge needle.

  1. Insert the needle at a 45° angle into the subcutaneous space in the back skin of the mouse, bevel up, by sliding the needle almost parallel along the skin in the base of the tent.
  2. Carefully inject the mouse with 50 μl of the inoculum.

     1. The bevel should be turned up, and the inoculum should be a visible pouch under the skin that contains the injected fluid. Check for any bleeding or leakage (which could lead to decreased bacterial numbers in the skin) and may indicate the needle was more in-depth than the intended subcutaneous tissue.
• 11Move mice to a warm environment to recover from anesthesia and return animals to the cage.

Monitoring lesion development

• 12Start measuring abscess and lesion development (dermonecrosis) 24 h after infection and then every other day for 10 days post-infection (Fig. 1).
• 13Using a caliper (Mitutoyo), measure the length (L), and width (W) of the lesion and apply the values to the standard formula for the area: (A = [π/2] × length × width) (Becker et al., 2014).
• 14Euthanize the animals 10 days post-infection using methods allowed by the local IACUC.

Alternative protocol 1: Murine Tape Stripping Skin Infection Model

In this model, infection is established by disrupting the skin barrier with partial removal of the epidermal layer using tape stripping and a subsequent application of S. aureus (Kugelberg, 2015). In this model, a luminescent strain with chromosomally expressed luciferase is used to visualize infection progression in IVIS imaging. Traditionally, this method has been used on the back of mice, particularly between the shoulder blades (Patrick et al., 2020). The protocol described here differs by relocating the infection area to the inner epithelial layer of each ear, which facilitates imaging studies.

Reagents and Solutions

• PBS

• Isoflurane

• Puralube® Vet Ointment

• Bacterial Inoculum (See Support Protocol 1)

Equipment

• 1.5 mL Eppendorf tubes

• 10 µL pipette

• 10 µL pipette tips

• Tensoplast® Adhesive Bandage

• Adroit® Heat Therapy Pump

• Scissors

• Permanent marker

• PerkinElmer IVIS Spectrum (In Vivo Imaging System)

Procedure

Tape Strip preparation

• 1Cut Tensoplast into 2 × 5 cm strips.
• 2Save strips until infection.

Tape Striping Skin Infection

• 3Label mice using permanent marker tallies on the tail.
• 4Weigh and record the weight of mice.
• 5Place heating pads in the hood and connect to the Adroit heating pump in preparation for anesthetized mice.
• 6Anesthetize mice using Isoflurane according to machine instructions.
• 7Place Tensoplast® strips to the inner ear and remove rapidly to eliminate hair and form a wound. Continue until the skin looks irritated and lacks hair (approximately 6–8 strips).
• 8Place 5 µL droplet of bacterial inoculum (approximately 10⁷ CFU) on the wound using a 10 µL pipette and 10 µL pipette tips.
• 9Allow the droplet to dry thoroughly.
• 10Place infected mice back in the cage.
• 11Place the cages on a heating pad following anesthetization and monitor mice until they gain consciousness post-anesthesia. This will usually take 2–3 hr.
• 12Image mice using IVIS. Follow instrument instructions to detect bioluminescence intensity.
• 13Monitor mice twice a day during infection, making sure to weigh the mice once per day. Moribund mice should be humanly euthanized by CO₂ inhalation.

Basic Protocol 2: Sample collection to determine skin structure, production of inflammatory mediators, and bacterial load.

Materials

• 8–10 mm punch biopsy tool

• Tweezers

• Scissors

• 70% ethanol

• Tryptic Soy Broth (TSB) (See Support Protocol 2)

• Petri dishes containing Tryptic Soy Agar (TSA) (See Support Protocol 2).

• 1.5 mL Microcentrifuge tubes.

• Aerosol-resistant tips for micropipettes (20, 200, and 1000 μL tips).

• 5, 10, and 25 mL pipettes.

• Ice

  1. Place euthanized (CO₂ chamber asphyxiation method), infected mouse dorsal side up on a clean paper towel.
  2. Spray the infection area lightly with 70% ethanol.
  3. Using a skin punch biopsy tool, cut the infected skin around the lesion, and use the tweezers to pull the biopsy straight up from the back of the mouse. Place each sample in a 1.5 mL microcentrifuge tube on ice.
  4. After collection, biopsies can be cut into two to four separate pieces for individually examining bacterial burden and determining levels of soluble mediators, RNA, and histological analysis as described below.
  5. For histological analysis, place the biopsy tissue section in appropriate fixative (PFA, acetone, etc.) for 24 h and move the tissue to 70% EtOH until tissue processing.
  6. For RNA, protein, or soluble mediators by ELISA, weight tissue sample and place in 200–400 μL of appropriate lysis buffer depending on analysis you plan to run.

     1. For protein and soluble mediator analysis (ELISA), RIPA buffer (Thermo Fisher Scientific, cat. no. 89900) plus protease inhibitors (Thermo Fisher Scientific, cat no. 78430) can be used.
     2. For RNA analysis, place the tissues in RNeasy Lysis Buffer (Qiagen, cat no. 79216).
  7. Homogenize biopsies in the respective lysis buffer (see above) using either a pestle or homogenizer before spinning at 12,000 RCF for 5 minutes at 4°C to remove tissue debris.
  8. Transfer the supernatant to a fresh tube, that is now ready for processing or analysis (Western Blotting, ELISA, RNA isolation). Divide final analysis results by the recorded tissue weight to present results per milligram of harvested tissue.
  9. To determine bacterial burden, first weight tissue sample and then place it in a 1.5 mL microcentrifuge tube containing 200 μL TSB (see Support Protocol 2). The biopsies are then homogenized with a pestle (3.5 in.) or homogenizer (Bullet Blender).
  10. Vortex each homogenized sample in TSB for 10–20 seconds before performing serial dilutions (undiluted, 1:100, and 1:1000) of the homogenate in TSB.
  11. Pipet 10–100 μL of each serial dilution onto the Petri dishes containing TSA (See Support Protocol 2). Spread the samples with an inoculation loop until an even coat of the solution is observed.
  12. Incubate the plates for 18–24 h at 37°C and count the number of colonies.
  13. Determine the number of colonies on each plate with fewer than 300 colonies. CFU/mL can be quantified as: (# colonies/volume plated (mL)) x Final Dilution Factor = CFU/mL. To correct for variance in the amount of biopsy tissue collected, divide the final results by the weight of the biopsy, and express the data as CFU/mg of tissue. Bacterial number values should range between 10⁷–10⁹ CFU/mg of tissue at day 10 after infection with 3–5 10⁶ CFU (See Basic Protocol 1).

Basic Protocol 3: Murine model of post-traumatic Staphylococcus aureus osteomyelitis

Introduction

The purpose of this protocol is to experimentally model S. aureus osteomyelitis. Osteomyelitis is one of the most common manifestations of the invasive staphylococcal disease, but treatment is notoriously difficult and involves prolonged antimicrobial therapy coupled with surgical debridement (Brandt et al., 2018; Hatzenbuehler & Pulling, 2011; Rao et al., 2011). The therapeutic recalcitrance of osteomyelitis reflects widespread antibiotic resistance in S. aureus and pathologic changes in bone remodeling that may limit antibiotic therapy and antibacterial immune responses (Brandt et al., 2018; Cassat et al., 2013; Putnam et al., 2019). By modeling osteomyelitis in a genetically tractable organism, the underlying host and pathogen contributions to disease can be better understood. Outcomes of this protocol can include the enumeration of bacterial burdens in bone, surrounding tissues, or other organ systems, histopathology, and radiographic analyses to determine how infection impacts bone architecture.

Reagents and Solution

• C57BL/6 mice, 6 to 10 weeks old, male or female (The Jackson Laboratory)

• Isoflurane

• O₂ Source

• 70% ethanol

• Bacterial inoculum (See Support Protocol 1)

• Depilatory cream (Nair®).

• Buprenex HCL

• 10% w/v Povidone-iodine.

• 70% Ethanol.

• Ophthalmic ointment

Equipment

• Animal clippers (ProClip clippers equipped with size 40 carbon-infused steel blade).

• ½ inch 30-gauge beveled needles

• 21-gauge PrecisionGlide needles

• Vicryl and Ethilon sutures

• 1-ml syringes

• 0.5-ml U-100 28G Insulin Syringe

• Isoflurane induction chamber with nose cone attachment

• Forceps.

• Deltaphase® isothermal pad

• Sterile cotton gauze pad.

• No. 10 surgical blades attached to a No. 3 scalpel handle

• Germinator™ 500 dry sterilizer

• Bullet blender®

Prepare the surgical site

• 1Weigh each mouse to be subjected to experimental osteomyelitis. This weight measurement will enable accurate dosing of analgesics and establish a baseline for postoperative weight loss monitoring.

  Our laboratory typically uses C57BL/6J mice, given the wealth of genetically modified strains available on the C57BL/6 background. Similar pathology and bacterial burdens have also been documented following FVB/NJ and BALB/cJ mice infection.

• 2Prepare a laminar flow biosafety cabinet for the surgical procedure by disinfecting all surfaces and establishing dedicated areas for both the surgical preparation and the sterile procedure.
• 3Induce anesthesia by transferring one mouse to an induction chamber and administering 3% isoflurane.

  An appropriate plane of anesthesia should be confirmed by monitoring respiratory rate, reaction to stimuli such as toe pinch, or other suitable maneuvers.

• 4Transfer anesthetized mouse from the induction chamber to a nose cone and maintain anesthesia by administering 1.5% isoflurane.
• 5Administer pre-operative analgesia (Buprenex HCL, 0.05 – 0.1 mg/kg) via subcutaneous injection using a 0.5-ml U-100 28G Insulin Syringe.
• 6Using a hair trimmer, shave the entire hindlimb and flank on either the left or right side of the mouse.
• 7Disinfect the surgical site and surrounding tissues by scrubbing with a 10% povidone-iodine prep pad and allowing the solution to dry. Repeat this process once.
• 8Complete surgical site disinfection by scrubbing with one alcohol prep pad.
• 9Move the mouse into the surgical field and place it on top of a pre-warmed (37°C) Deltaphase® isothermal pad that has been covered with a sterile towel.

  Ensure that the prepared surgical site is not touched from this point forward without sterile technique.

• 10Drape the mouse with an additional sterile towel such that only that surgical site is exposed.

  Towels can be pre-cut down the middle of the long axis to facilitate draping of the surgical site. These cut towels should be sterilized before surgery.

Inoculation of the intramedullary canal with S. aureus

• 11Confirm sufficient depth of anesthesia and proper placement of nose cone before surgery.
• 12Create an approximately 9-mm incision in the skin overlying the mid-femur lateral aspect using a No. 10 surgical blade attached to a No. 3 scalpel handle. The incision should extend from the proximal to distal metaphysis to cover the middle two-thirds of the femur (Fig 2).
• 13Following the course of the skin incision, incise the muscle and fasciae directly overlying the femur.
• 14Using Adson forceps, bluntly dissect the muscle and fasciae to expose the mid-femur.
• 15Maintain exposure of the mid femur by retracting muscle and fascia with the Adson forceps. Using a 21-gauge PrecisionGlide needle, create a unicortical bone defect in the mid-femur by trephination.

  The unicortical defect should be created in the mid-femur anterolateral aspect, where the bone surface is often flatter. Ensure that the unicortical defect does not compromise the endosteal surface of the opposite bone cortex.

• 16Facilitate hemostasis at the unicortical defect site by applying pressure with a sterile cotton gauze sponge for approximately 15 seconds.
• 17Using a micro pipettor with an attached sterile gel-loading pipette tip, inoculate 2 microliters of the S. aureus inoculum directly through the unicortical bone defect and into the intramedullary canal (Fig 2).
• 18Approximate muscle and fasciae are overlying the inoculation site with one or more 5–0 Vicryl sutures using needle holder and Adson forceps.
• 19Close the skin incision with two or more 5–0 Ethilon sutures.
• 20Remove the sterile drape and carefully return the mouse to a new cage that has been placed on two pre-warmed (37°C) Deltaphase® isothermal pads.
• 21Monitor respiratory rate and spontaneous movements until the mouse has recovered from anesthesia.
• 22Sterilize the surgical instruments between each mouse using a Germinator™ 500 dry sterilizer or equivalent.

  A new surgical blade, sterile drape, sterile gauze pads, gel-loading pipette tip, 21-gauge PrecisionGlide needle, and Vicryl and Ethilon sutures should be used for each mouse.

Postoperative care

• 23Administer analgesia (Buprenex HCL, 0.05 – 0.1 mg/kg, via subcutaneous injection) every 12 hours for the first 48 hours and then every 12 hours as needed for pain for the duration of the experiment.

  Signs indicative of pain include, but are not limited to, decreased mobility, altered gait, hunched posture, excessive grooming of surgical site, and weight loss.

• 24Weigh each mouse at least once daily, or at the frequency suggested by the institutional animal care and use committee.
• 25Monitor mice for humane endpoints and euthanize those mice that meet humane endpoints.

  Humane endpoints should be predetermined in consultation with veterinary staff and the institutional animal care and use committee. Examples of humane endpoints include weight loss greater than 20% of initial body weight, impaired mobility despite analgesia administration, wound dehiscence, and secondary wound infection signs.

Euthanasia, tissue harvest, and bacterial enumeration

• 26At the experimental endpoint, humanely euthanize the infected mice per institutional guidelines.

  Our laboratory typically assesses experimental outcomes at up to 14 days post-infection, a duration at which bacterial burdens are frequently stable, and there is significant induction of pathologic bone remodeling.

• 27Disinfect the euthanized mouse by spraying with 70% ethanol solution.
• 28Using Adson forceps and a surgical blade, carefully expose the femur by incising through the skin and soft tissues.
• 29Remove the femur by transecting the patellar tendon with surgical scissors and disarticulating the head of the femur from the hip joint with either surgical scissors or a surgical blade.
• 30Remove soft tissue from the disarticulated femur using sterile technique and place the femur in a Bullet blender® Navy Bead Lysis Kit 1.5mL microcentrifuge tube. Keep the tube on ice until the femur is homogenized.
• 31Add 500 μL of sterile PBS to the Bullet blender® tube. Homogenize the femur by performing three consecutive 5-min runs of the Bullet Blender® machine on speed setting 12.

  Our laboratory keeps the Bullet blender® machine in a cold room to conduct all homogenization steps at 4°C.

• 32Enumerate bacterial burdens from each homogenized femur by serially diluting the homogenate in sterile PBS and plating at limiting dilution on tryptic soy agar. Incubate TSA plates overnight at 37°C.

Basic Protocol 4: Intravenous Infection of the Retro-orbital Sinus

Introduction:

Invasive S. aureus infection is associated with systemic infection and abscess lesions in virtually all organs (Cheng et al., 2009; Lowy, 1998). S. aureus can be found in many organs in murine models of systemic infection, and abscesses can be detected in musculoskeletal, brain, lung, heart, liver, spleen, and kidney tissues (Cheng et al., 2011). Therefore, these organs have been extensively used to study infection dynamics and provide valuable information regarding both the host immune response and in vivo microbial pathogenesis. Here, we describe a model of retro-orbital injection of the venous sinus that is easily mastered and a humane approach to intravascular injections that can reliably deliver up to 150 μL to generate systemic infections using S. aureus.

Reagents and Solutions

• PBS

• Avertin (See Support Protocol 2)

• Puralube® Vet Ointment

• Tryptic Soy agar (TSA; 2% agar) (See Support Protocol 2)

• Tryptic Soy broth (TSB) (See Support Protocol 2)

• Bacterial Inoculum (See Support Protocol 1)

Equipment

• 15 mL conical tubes

• 50 mL conical tubes

• 60 mL syringe

• 0.45 μm syringe filter

• 500 μL Insulin syringe

• Cotton tipped applicator

• TSA plates (See Support Protocol 2)

Procedure

Retro-orbital Infection

1. Anesthetize mice by injecting Avertin (312.5–375 mg/kg) into the peritoneum. For a 20 g mouse, this would be 250–300 μL

2. Load 100 μL of bacterial inoculum into a 500 μL insulin syringe (1 per mouse).

3. Weigh and record the weight of the mice.

4. Retro-orbitally inject 100 μL of the S. aureus inoculum (1–2×10⁷ CFU).

5. Apply Puralube® Vet Ointment to the eye immediately after injection using a sterile cotton-tipped applicator. Place infected mice into a clean cage.

6. Place the cages on a heating pad following anesthetization and monitor mice until they gain consciousness post-anesthesia. This will usually take 2–3 hr.

7. Monitor mice twice a day during infection, making sure to weigh the mice once per day. Moribund mice should be humanly euthanized by CO₂ inhalation.

Be sure to mark the mice after the first weighing to keep track of individual mice in a single cage. This can be done by ear punch or marking the tails using permanent markers.

Support Protocol 1: Preparation of the bacterial inoculum.

• Frozen aliquot of S. aureus containing at least 10⁶ CFUs. Here, most of the protocols were tested using the MRSA USA 300 LAC strain

• Tryptic Soy Broth (TSB)

• Sterile phosphate-buffered saline (PBS)

• 1.5 mL Microcentrifuge tubes.

• Aerosol-resistant tips for micropipettes (20, 200, and 1000 μL tips).

• 5, 10, and 25 mL pipettes.

• 50mL conical Tubes

• Orbital Shaker at 200 RPM and 37°C

• Spectrophotometer (reading at OD600)

• Refrigerated centrifuge

Proper culturing and preparation of S. aureus are required to produce reliable and consistent in vivo infection models. S. aureus in the log phase of growth is preferable for this infection model, and before this protocol, you will need to generate a growth curve of your S. aureus strain of choice based on the OD₆₀₀ absorbance readings of your culture. S. aureus must also be washed before resuspension and injection to remove any number of the toxins produced by S. aureus during in vitro culture.

• 1Aliquot 10 mL of TSB in a 50 mL conical tube.
• 2Inoculate the overnight culture tube with an aliquot of frozen S. aureus. Leave the caps of the tubes slightly loose to allow for air to enter the conical.
• 3Place tubes in the orbital shaker overnight at 37C and 200 RPM.
• 4Dilute the overnight TSB culture 1:100 into 10 mL of fresh TSB and culture an additional 3 hours at 37°C with 180 r.p.m. orbital shaking to allow the overnight culture to re-enter the log phase of growth.
• 5Measure the OD600 and compare it to the growth chart to see the concentration of S. aureus in your subculture and ensure it reaches the log phase of growth.
• 6Harvest bacterial cells from the 3-hour culture by centrifugation at 8,000 x g for 8 min 4°C.
• 7Resuspend pellet in 1 mL PBS and wash the bacterial cells 2 times as above.
• 8Resuspend your final bacterial pellet in the appropriate amount of PBS to reach your desired concentration for infection.

The optical density corresponding to the desired inoculum concentration should be determined empirically by plating a dilution series on tryptic soy agar to enumerate bacterial colony-forming units. Our laboratories routinely use inoculum of 1×10⁴ to 3×10⁷ colony forming units depending on the biologic question under study, models of infection, the fitness and virulence of the infecting strain, and the host genetic background.

Be aware that how the 1:100 sub-culture is performed can have a significant effect on virulence. In our hands, bacteria from sub-cultures incubated at 37 °C in a shaking incubator tend to be more virulent than equivalent amounts of bacteria from sub-cultures incubated at 37 °C using a roller drum despite similar CFUs.

When quantifying the OD₆₀₀ and diluting S. aureus, make sure to vigorously vortex the bacteria as S. aureus tends to clump in the tubes. This will create more accurate readings and dilutions.

• 9Once resuspended to the desired concentration, transfer the inoculum to 1.5 mL centrifuge tubes and place them on ice until ready for inoculation.

Support Protocol 2: Preparation of reagents and solutions:

Tryptic soy broth (TSB - Soybean-Casein Digest Medium).

• 1Weight 30 g of TSB powder and dissolve in 1000 ml distilled water.
• 2Place the bottle on a magnetic stirrer to mix under gentle heat.
• 3Aliquot 100 ml of the medium to an autoclavable container.
• 4After aliquot, place the container into an autoclave at 121°C for 15 minutes.
• 5Remove the flask and make sure the cap of the tube is closed tightly.
• 6Fully cool autoclaved tubes at room temperature before placing stock in 4°C refrigerator and avoid light. Pre-warm the medium to room temperature before usage.

Trypic Soy Agar (TSA) Plates:

• 7Prepare media as mentioned for TSB, add 15 g of Agar and autoclave.
• 8In a biosafety cabinet, cool media to ~ 45–50°C, mix and dispense quickly into sterile Petri dishes (20–25 ml per 100 × 15–mm plate).
• 9Allow to solidify with the lid half off, then dry thoroughly until no visible moisture remains on the agar surface.
• 10Place plates in a plastic sleeve and seal. Store upside down up at 4°C.
• 11Let the dry plates at room temperature before use.

Avertin preparation:

• 12Add 1 g 2,2,2-tribromoethanol (Avertin) to 1 mL tert-amyl alcohol in a 1.5 mL Eppendorf tube. Rock at room temperature in the dark. Every 30 min, vortex the tube and return to the rocker. Avertin will take 1.5–2.5 h to go into solution.
• 13Once entirely in solution, dilute the Avertin 1:40 into warm sterile PBS.
• 14Sterile filter the Avertin and load enough 500 μL insulin syringes (1 per mouse) with 400 μL of diluted Avertin. Keep the syringes at room temperature in the dark until the infection.

After the 1:40 dilution of Avertin into PBS, the Avertin may partially precipitate because the PBS is too cold. If this occurs, place the Avertin tube at 37 °C in the dark and vortex every 10 min. Once the Avertin is entirely in solution, proceed with the sterile filtering.

4. Background information.

S. aureus infections caused by methicillin-resistant or susceptible strains are an enormous burden to both infected individuals and on the healthcare system worldwide. S. aureus can cause many disease manifestations, ranging from superficial, suppurative, and subcutaneous skin infections to invasive infections, including pneumonia, sepsis, septic arthritis, endocarditis, and osteomyelitis. Infections can become persistent, causing abscess formation (Brandt et al., 2018). The progression of the disease is often opportunistic and affects both immunocompromised and healthy individuals. Also, no vaccine is available for humans. Therefore, a better understanding of the disease pathogenesis, immune response, and vaccination protocols are needed. Animal models of S. aureus infection are a critical tool used to understand both cellular and molecular characteristics of the disease to develop effective therapeutics against these hard-to-treat infections.

Murine models of staphylococcal infections are useful to provide experimental evidence of the microbial pathogenesis, such as the discovery of virulence factors, understand mechanisms of tissue injury triggered by both bacterial and host factors, and development of immunity in both vaccine models and disease recurrency. Mouse models are the gold standard animal to study S. aureus infection due 1) to the surplus of reagents available (mouse strains, antibodies, inhibitors), 2) maintenance costs, 3) short gestation, 4) the mouse is readily amenable to drug and/or gene therapies and 5) overall similarities regarding immune response and physiology with the human body. However, the relevance of mice for S. aureus infection models has often been interrogated because experimental infection of mice with S. aureus clinical isolates usually requires high bacterial doses to cause pathologies. Although it has been suggested that S. aureus can colonize both wild rodents and laboratory mice, the pathogen requires genetic adaptation, and a few clonal complexes (CC) 1, 15, and 88 are also found in humans. These strains are also virulent in mice models of sepsis, but their pathogenesis in different mouse models has not yet been examined in detail. Mice have been used as a primary model to study S. aureus bacteremia, septic arthritis, meningitis, neonatal sepsis, pneumonia, osteomyelitis, subcutaneous, and superficial skin infections, to name a few. Using these models, it has been clear to investigators that S. aureus can cause a myriad of diseases that are also present in humans (despite the higher doses of the bacteria required to infect animals); murine models have generated critical findings regarding the genetic determinants of resistance or susceptibility to infections, the role of both innate and adaptive immune cells in the establishment, severity, and chronicity of infections, mechanisms of pathogen survival, virulence factors and toxins required to cause disease.

5. Time considerations

Subcutaneous infection:

Hair removal takes about 10 min/animal. For the infection, after preparation of the inoculum, the injection should take about 5–10 min/animal. Harvesting skin biopsies requires ~ 10 min. Single-cell suspension and bacterial enumeration should take around 60 min. Imaging the bacterial using an IVIS imaging system should take approximately about 10 min/animal.

Tape stripping skin infection:

After preparing the tapes and bacterial inoculum, the infection should take around 30 min/mouse.

Osteomyelitis model:

Depending on the experience of the investigator, the surgical procedure to induce osteomyelitis takes 10–20 minutes per mouse. Harvesting of infected femurs requires approximately 10 minutes per mouse.

Intravenous infection model:

Injecting mice with retroorbital inoculation requires training. After obtaining proper training, the injection should take about 5 min per mouse

6. Critical Parameters and Troubleshooting.

General potential pitfalls in S. aureus infection models:

A few considerations should be cogitated when planning infection experiments with S. aureus. 1) Animal age, weight, and strain should be matched to ensure reproducibility and robust power analysis. 2) Different strains of S. aureus express different amounts of toxins and virulence factors that could induce various pathologies. Furthermore, consistency in the generation of frozen stocks and bacterial growth is necessary to ensure reproducibility. 3) Some infection models require a high bacterial number to cause disease, and therefore, it may not mimic pathogenesis in humans, where lower bacterial numbers can induce pathology. 4) Mice should always be injected with PBS or vehicle control as an uninfected control. 5) It is highly recommended to co-house animals for the adaptation of different microbiotas.

Skin infection:

Since the immune response in the epidermis and dermis is somewhat different, a big challenge in the subcutaneous infection is the injection depth and the potential leakage of the injected bacteria. The sample collection can also raise confounding results. To avoid excluding areas of the abscess and/or rich in bacteria, our laboratory uses an 8 mm biopsy punch (Acuderm, Inc), and we split the sample into two pieces for the detection of bacterial loads and histology (for the determination of abscess formation and cell recruitment). By splitting the biopsy into two parts, we have enough material to determine critical endpoints without compromising the sample collection and interexperimental variability.

Osteomyelitis:

During or following surgery, mice can sustain pathologic fractures, which are identified by impaired mobility despite adequate analgesic administration. These mice are removed from the experiment.

Systemic infection:

The kidneys are usually the first target organ during systemic infection with S. aureus and are often used as a surrogate marker to determine disease severity. However, S. aureus can infect multiple organs to different degrees, which should be considered for acute and chronic systemic disseminated and G.I. disseminated models.

7. Anticipated results

Skin infection:

Within 24 hours after infection, a small abscess is observed due to the inflammatory response induced by the bacteria. The abscess increases substantially by day 3 (30–100 mm²), and a lesion (dermonecrosis) is formed around day 4–7, followed by resolution of the abscess and lesion size by days 8–10 (Fig. 1). Depending on the S. aureus strain tested and its production of secreted α-hemolysin (α-toxin), subcutaneous skin lesions are associated with superficial dermonecrosis, which heals at a similar rate as the resolution of subcutaneous abscess lesions. We typically observe an increase in 1–2 logs from the ~3×10⁶ CFU inoculum after 12–24 hours post-infection, and this bacterial remains the same up to day 4–6 post-infection. We then observe a steady decline in bacterial loads at day 7–10 after infection. After 10 days of infection, we still detect S. aureus in the skin, even when the dermonecrosis is absent. Detection of bioluminescent bacteria in the tape stripping method can be done daily using the IVIS imaging system.

Osteomyelitis:

Our laboratory typically performs histology at days 4, 7, and 14 after infection, followed by microCT imaging (to determine the bone loss in both cortical and trabecular bone). For the bacterial loads, we typically see an initial increase of 1–2 logs from the 10⁶ inoculums over the first 24–48hours, followed by a decline back to 10⁶ around day 5–7. In C57BL/6 mice, these burdens persist at about 10⁶ for the remainder of the experiment.

Systemic infection:

S. aureus can be detected in the kidney, spleen, and liver as early as 12 hours following Injection of 5 ×10⁷ CFU via retroorbital inoculation (Perry et al., 2019). By day 2–4, we can detect multiple abscesses in the liver and kidney in tissue sections using stained with H&E. The abscesses should not increase in size, but numbers in different organs. Increased mortality can be observed around day 3–5, and around 30% of mice survive at day 10 post-infection. The bacterial load in the kidney increases in the first 24 hours and peaks at 4 days post-infection.