Rabbit Models for Infectious Diseases Caused by Staphylococcus aureus

Abstract

Staphylococcus aureus (S. aureus) is a disreputable symbiotic bacterium that is responsible for a range of diseases, including life-threatening pneumonia, endocarditis, septicemia, and others, which has led to an immense loss in both public health and economy, imposing a significant burden on society. To investigate the pathogenic mechanism of S. aureus and develop new treatment methods for infectious diseases caused by S. aureus, various rabbit models have been developed to simulate different infections by S. aureus, such as pneumonia models, meningitis models, and endocarditis models, etc. In this review, we summarized the application of rabbit models in S. aureus-induced infectious diseases.

1. Introduction

Staphylococcus aureus (S. aureus) is a Gram-positive bacterium and appears grape-like under the microscope. The human body is a repository of S. aureus, with an estimated 30% of the population asymptomatically colonized by the bacterium [1]. S. aureus commonly inhabits the nasal cavity, skin, and gastrointestinal tract [2]. Meanwhile, S. aureus acts as an opportunistic pathogen, capable of causing a range of diseases worldwide, including mild skin and soft tissue infections such as impetigo and uncomplicated cellulitis, as well as potentially life-threatening diseases like infective endocarditis, osteoarticular infections and toxic shock syndrome [3]. The pathogenesis of infections is primarily attributed to numerous surface and secreted virulence factors of S. aureus [4].

As a major culprit behind human infectious diseases [5], S. aureus has led to substantial morbidity and mortality. Antibiotics are routinely employed for the treatment of S. aureus infections. However, due to the overuse of antibiotics and the remarkable adaptability of S. aureus, drug-resistant strains have emerged, among which methicillin-resistant S. aureus (MRSA) poses the most substantial threat. A study in China revealed that the total hospital cost and antibiotic cost for MRSA patients has increased by an average of USD 3220 and USD 672, respectively, compared to methicillin-sensitive S. aureus (MSSA) patients [6]. In addition to conventional antibiotic therapy, various alternative approaches for the treatment of MRSA have been explored [7], including the utilization of phytochemicals, natural compounds such as garlic and ginger, and bacteriophage therapy. However, the situation remains critical.

Novel strategies are urgently required to combat drug-resistant isolates and control the spread and infections of S. aureus. A thorough understanding of the pathogenesis of S. aureus is indispensable, which requires the assistance of appropriate animal models that accurately simulate human infections in pre-clinical studies. While mice are widely used in the study of disease pathogenesis because of their low cost [8], they are not natural hosts for S. aureus [9], and rodent models are flawed in many aspects for the study of human development and diseases [10]. Therefore, the conventional mouse model may not be the most suitable choice for studying S. aureus infections. In contrast, the larger size of the rabbit makes it possible to monitor non-fatal physiological changes [10], and the immune and cardiovascular systems of rabbits closely resemble those of humans in response to S. aureus infections [8]. Furthermore, rabbit models have been employed in studying infectious diseases caused by S. aureus for over two centuries [11]. To elucidate the mechanisms of disease onset and progression, and explore potential treatment options, various rabbit models of S. aureus infections, including pneumonia model, osteomyelitis model, endocarditis model and so on (Figure 1), have been established. This work conducted a systematic literature search in PubMed and Web of Science databases, employing the following parameters: (1) keywords including “infectious diseases” (e.g., pneumonia, endocarditis, keratitis, meningitis, osteomyelitis), “rabbit model” and “Staphylococcus aureus”; (2) exclusion of articles published before 2000; and (3) exclusion of studies focusing on rabbit-specific diseases, and summarized the latest advances of the application of rabbit models in S. aureus-induced infectious diseases.

2. Pneumonia

S. aureus is a significant pathogen for both hospital-associated pneumonia (HAP) and community-associated pneumonia (CAP) [3], and pneumonia induced by S. aureus caused a high mortality, especially CAP with its mortality rate ranging from 30–70% [12].

Numerous rabbit models of pneumonia induced by S. aureus have been reported. For instance, with the assistance of bronchoscopy, Kong et al. [12] inoculated S. aureus into the first bronchiole of the lateral bronchiole of the basal segment of the right lower lobe of the rabbit lung to establish a rabbit model of pneumonia. Strandberg et al. [13] performed a 3-mm tracheal incision in the rabbit’ neck, and subsequently inserted a polyethylene catheter into the left bronchus for S. aureus inoculation, while Paharik et al. [14] opted for a midline abdominal incision. Histological examination revealed massive hemorrhage in the lungs of the model rabbits in all three studies.

Several studies have investigated the underlying mechanisms of pneumonia caused by S. aureus in rabbit models. For example, Diep et al. [15] found that Panton-Valentine leucocidin (PVL) relies on the recruitment and lysis of polymorphonuclear leukocytes to cause lung infection and injury in rabbit pneumonia model. Notably, the majority of S. aureus-induced pneumonia cases are caused by PVL-negative bacteria [16,17]. When producing elevated concentrations of the Hlg CB component, strain PEN (PVL⁻) [18] exhibited similar virulence to PVL⁺ strains in rabbit pneumonia model. A protease called Spl [14], that is unique to S. aureus, also contributes to the development of pneumonia, pathological findings revealed hemorrhage in both lungs of rabbits infected with wild-type (WT) S. aureus, whereas only one lung in rabbits infected with the mutant bacteria (ΔSpl::erm).

Several studies utilized S. aureus-induced rabbit pneumonia models to evaluate the new treatment strategies. For example, α-Hemolysin (Hla) was clinically regarded as the target to treat or prevent S. aureus pneumonia [19,20,21,22], Diep et al. [11] employed MEDI4893*, a monoclonal antibody targeting Hla, to conduct passive immunization in rabbits infected with USA100 strains, which exclusively produce Hla. The results showed that the survival rate of rabbits infected with USA100 was 100%.

The summary information of reported S. aureus pneumonia rabbit models is presented in

Table 1. The summary information of S. aureus-induced pneumonia models in rabbits.

Author	Rabbit Species	Dose	Strains	Inoculation Mode	Duration
Cruiser-Bertin [23]	New Zealand white rabbit	3 × 109 CFU/mL 0.5 ml	USA300	Inoculation via jugular catheter	50 h
Kong [12]	New Zealand white rabbit	1 × 1010 CFUs	ATCC 33591	Inoculation by bronchoscope	7 d
Strandberg [13]	Dutch belted rabbit	2 × 109 CFUs	USA200 USA400	Inoculation by tracheal intubation	7 d
Diep [24]	New Zealand white rabbit	5–6 × 109 CFUs	USA300 SF8300	Inoculation via pediatric endotracheal tube	36 h
Diep [15]	New Zealand white rabbit	2–3 × 1010 CFU/mL 1.5 mL	USA300 SF8300	Inoculation via pediatric endotracheal tube	48 h
Paharik [14]	Dutch belted rabbit	2 × 109 CFUs	USA300 LAC	Inoculation via ventral midline tracheal cannula	6 d
Pivard [18]	New Zealand white rabbit	9.49–9.61 log10 CFU/mL, 0.5 mL	USA300 ST80 PEN	Inoculation by intratracheal instillation	48 h
Diep [11]	New Zealand White outbred rabbits	Not provided	USA300 SF8300	Inoculation via pediatric endotracheal tube	96 h

, highlighting the similarities and discrepancies in terms of rabbit species, modes and doses of inoculation, duration of infections, and types of S. aureus strains. The inoculation modes encompassed direct tracheal intubation and incision tracheal intubation. The most frequently utilized rabbit species was the New Zealand white rabbit. The doses of S. aureus inoculation ranged from 10⁹ to 10¹⁰ colony forming units (CFUs). The range of infection duration varied from 36 h to 7 days, which may be related to different experimental purposes and procedures. For instance, Kong et al. [12] aimed to develop a model of severe pneumonia, and rabbits in the model group exhibited peak body temperature and minimum body weight on day 7. In the study by Strandberg et al. [13], prolonged monitoring for the development of respiratory distress and fatal disease was required, and day 7 was defined by the guidelines as the point at which the rabbit died or lost the ability to escape or self-correct.

3. Skin and Soft Tissue Infections

Skin and soft tissue infections (SSTIs) caused by S. aureus are a significant health concern. While most SSTIs are mild and self-limiting, complications can lead to fatal outcomes [25]. In 2019, SSTIs caused by S. aureus led to an all-cause, age-standardized mortality rate of 0.5 globally [26].

Several reported studies have discussed the establishment of rabbit SSTIs models. Le et al. [27] intradermally injected 120 μL of SF8300 at a concentration of 2.5 × 10¹⁰ to 3.0 × 10¹⁰ CFU/mL into the shaved and disinfected dorsal right lumbar spine skin of the rabbits by a 1-mL insulin syringe, the infection was maintained for seven days. This model effectively mimics severe human skin infections, and rabbits developed severe skin necrotizing ulcers. Nevertheless, in the study of Li et al. [28], which also utilized intradermal inoculation, rabbits did not present the same performance with the dose of inoculation 5 × 10⁸ CFUs, consistent with the experimental results of Le et al. Rabbit models require high doses of S. aureus to cause reproducible disease, at least 1 × 10⁸ CFUs [29]. However, human skin infections may not need such large doses, which may obscure strain virulence and bacteria-host interactions [30]. To develop better models, Malachowa et al. [30] developed a low-inoculum rabbit model through subcutaneous injection of USA300 strain with dltB gene harboring in rabbit S. aureus, and induction of SSTIs rabbit model with this strain requires only 1 × 10⁶ CFUs. In the study by Muñoz-Silvestre et al. [8], rabbits were infected by intradermal inoculation with 300 CFUs of the rabbitized strain FdltB^r, and at 1 day postinfection (dpi), erythema appeared at the inoculation site, followed by nodules and necrosis. The difference in inoculation dose between the two studies may be due to the discrepancies in inoculation methods and bacterial lineage. Additionally, So-In et al. [31] successfully induced the rabbit dermatitis model by using a sterile blade firstly to create a wound at the designated infection site after the rabbits were anesthetized, and then inoculating S. aureus into the wound for a 48-h period.

PVL and Hla are important virulence factors responsible for skin infections. In the research of Chi et al. [32], rabbits infected with PVL-positive strains exhibited more extensive lesions and severe inflammatory responses compared to those infected with PVL-negative strains; in the rabbit model developed by Le et al. [27], it was observed that immunoprevention with MEDI4893* significantly mitigated the severity of the disease caused by the USA300 WT strain.

Table 2. The summary information of S. aureus-induced skin and soft tissue infection models in rabbits.

Author	Rabbit Species	Strains	Dose	Inoculation Mode	Duration
Le [27]	New Zealand White rabbit	SF8300	2.5–3.0 × 1010 CFU/mL 0.12 mL	Inoculation by intradermal injection	7 d
Li [28]	New Zealand White rabbit	SF8300	5 × 108 CFUs	Inoculation via intradermal injection	14 d
Malachowa [33]	New Zealand White rabbit	USA300 LAC	5 × 108 CFUs	Inoculation via subcutaneous injection	14 d
Malachowa [30]	New Zealand White rabbit	ST121 dltB/Δrot	1 × 106 CFUs	Inoculation via subcutaneous injection	14 d
Kobayashi [29]	New Zealand White rabbit	USA300	5 × 108 CFUs	Inoculation via subcutaneous injection	14 d
Muñoz-Silvestr [8]	Albino hybrid rabbit	FdltBr strains	300 CFUs	Inoculation by Intradermal injection	7 d
So-In [31]	New Zealand White rabbit	ATCC 6538	1 × 106 CFUs	Inoculation by applying S. aureus to skin wound	7 d

summarized the construction of various reported rabbit SSTIs models, which described that New Zealand White rabbits were frequently used species, the inoculated doses were ranging from 10⁶ to 10⁹ CFUs except Muñoz-Silvestre’s [8]. The mode of inoculation can be broadly divided into two categories, subcutaneous and intradermal, and the duration of infection was concentrated at 7 and 14 days.

4. Osteomyelitis

Osteomyelitis is a severe infectious disease that can lead to a detrimental outcome like pyogenic arthritis and abnormal bone remodeling without appropriate treatment [34]. The clinical presentation of osteomyelitis demonstrates considerable heterogeneity, with lacking a universally accepted classification system. Nevertheless, the Waldvogel system and Cierny-Mader system remain extensively referenced in clinical practice, for offering key information about the disease [35] S. aureus is the primary pathogen responsible for osteomyelitis, approximately accounting for 60% of all cases of osteomyelitis [36]. Furthermore, infections caused by PVL-positive strains demonstrated more persistent clinical courses and exhibited accelerated progression [37].

The establishment of rabbit osteomyelitis models presented greater challenges compared to SSTIs and pneumonia models as the S. aureus needs to be inoculated directly into the bone marrow cavity. Here are several representative studies, in the report of Kishor et al. [38], a 2-cm incision was made lateral to the distal femur of the rabbits after the rabbits were anesthetized and disinfected to expose the metaphyseal. A single cortical defect measuring 5 mm in diameter was created with the aid of a hand drill, intramedullary inoculation was followed to infect rabbits. For the wound management, the incision was covered with a sterile bandage and stitch was made in the middle of the open area. Unlike Kishor, Xu et al. [39] made a 2-cm skin incision on the anterolateral surface of the rabbit’s right proximal tibia to expose part of the metaphyseal cortex, and used a 2-mm kirschner to create a hole, subsequently irrigated with saline and aspirated the bone marrow with an 18-gauge needle. Finally, 0.1 mL 5% sodium morrhuate and MRSA bacterial suspension were injected into the medullary cavity successively. Radiographic results showed that bone infection reactions, including osteolysis and osteonecrosis, were observed in both rabbit models. In the study of Crémieux et al. [37], non-surgical method was utilized. 0.1 mL of 3% sodium tetradecyl sulfate, 0.2 mL of inoculum and 0.1 mL of saline were percutaneously injected into the medullary cavity in sequence through the lateral side of the right tibial metaphyseal using an 18-gauge needle to infect rabbits.

Treatment of osteomyelitis frequently necessitates long-term antibiotic therapy [40]. Rabbit osteomyelitis models have been used to discover novel treatment options for S. aureus, such as 1,8-Cineole [41], a component of essential oils, which was found to synergize with gentamicin and amoxicillin/clavulanic acid, greatly reducing the use of antibiotics. However, it is difficult to achieve effective bactericidal drug concentrations at the site of infection with conventional intravenous or oral administration methods. In rabbit models, the new drug delivery systems have been developed to achieve topical antibiotic treatment, for example, nanoparticle [42], hydroxyapatite cement [43], bone-like hydroxyapatite/poly amino acid [44] and synthetic calcium phosphate beads [45] have been utilized as carriers to load antibiotics. These carriers are biodegradable materials that can slowly release antibiotics, ensuring the concentration at the infection site sustained higher than the minimum inhibitory concentration (MIC).

Osteomyelitis models can be established through a variety of methods, broadly categorized into surgical and non-surgical approaches. Notably, there are distinct variations in wound management among surgical models. In some studies, the osteomyelitis model was induced by using S. aureus strains obtained from patients, and doses varied ranging from 10⁴ to 10⁹ CFUs. The summary information can be found in Table 3.

Table 3. The summary information of S. aureus-induced osteomyelitis models in rabbits.

Author	Rabbit Species	Strain and Source	Dose	Inoculation Mode	Wound Management
Kishor [38]	Rabbits	MRSA isolated from patients	5 × 106 CFU/mL 0.01 mL	Inoculation by intramedullary injection	Covered with sterile bandage
Jacqueline [42]	New Zealand rabbits	MRSA	1 × 108 CFUs	Intracavitary inoculation of the knee	Not provided
Joosten [43]	New Zealand rabbits	SCV-A22616/3 MRSA-W23, isolated from patients	3 × 106 CFUs	Inoculation by intramedullary injection through an opening	Closed with sutures
Zahar [46]	New Zealand rabbit	MRSA isolated from patients	1 × 108 CFUs	Inoculation by intramedullary injection through a bone deficit	Closed with sutures and nylon sutures
Xu [39]	New Zealand rabbit	ATCC 43300	1 × 106 CFU/mL 0.1 mL	Inoculation by intramedullary injection through a hole	Closed with 4/0 vicryl
Yan [44]	New Zealand rabbit	ATCC 25923	3 × 108 CFU/mL 0.2 mL	Inoculation by intramedullary injection through a hole	Closed with layered sutures
Crémieux [37]	New Zealand rabbit	USA300 LAC	8 × 105 CFUs 4 × 108 CFUs	Inoculation by intramedullary injection	Not provided
Hriouech [41]	New Zealand rabbit	MRSA isolated from patients	1 × 109 CFUs	Inoculation by intramedullary injection	Not provided
Muñoz [47]	New Zealand rabbit	S. aureus UASM-1	2 × 106 CFUs	Inoculation by marrow space injection	Sutured after bone restored
Amador [48]	New Zealand rabbit	MRSA obtained from blood cultures with an MIC of 1 g/mL vancomycin	1 × 109 CFUs	Inoculation by knee cavity injection	Not provided

Table 3. The summary information of S. aureus-induced osteomyelitis models in rabbits.

Author	Rabbit Species	Strain and Source	Dose	Inoculation Mode	Wound Management
Kishor [38]	Rabbits	MRSA isolated from patients	5 × 106 CFU/mL 0.01 mL	Inoculation by intramedullary injection	Covered with sterile bandage
Jacqueline [42]	New Zealand rabbits	MRSA	1 × 108 CFUs	Intracavitary inoculation of the knee	Not provided
Joosten [43]	New Zealand rabbits	SCV-A22616/3 MRSA-W23, isolated from patients	3 × 106 CFUs	Inoculation by intramedullary injection through an opening	Closed with sutures
Zahar [46]	New Zealand rabbit	MRSA isolated from patients	1 × 108 CFUs	Inoculation by intramedullary injection through a bone deficit	Closed with sutures and nylon sutures
Xu [39]	New Zealand rabbit	ATCC 43300	1 × 106 CFU/mL 0.1 mL	Inoculation by intramedullary injection through a hole	Closed with 4/0 vicryl
Yan [44]	New Zealand rabbit	ATCC 25923	3 × 108 CFU/mL 0.2 mL	Inoculation by intramedullary injection through a hole	Closed with layered sutures
Crémieux [37]	New Zealand rabbit	USA300 LAC	8 × 105 CFUs 4 × 108 CFUs	Inoculation by intramedullary injection	Not provided
Hriouech [41]	New Zealand rabbit	MRSA isolated from patients	1 × 109 CFUs	Inoculation by intramedullary injection	Not provided
Muñoz [47]	New Zealand rabbit	S. aureus UASM-1	2 × 106 CFUs	Inoculation by marrow space injection	Sutured after bone restored
Amador [48]	New Zealand rabbit	MRSA obtained from blood cultures with an MIC of 1 g/mL vancomycin	1 × 109 CFUs	Inoculation by knee cavity injection	Not provided

5. Keratitis

Keratitis, a type of corneal infection, can be caused by pathogenic microorganisms or non-infectious factors. Disruption of the corneal epithelial barrier facilitates bacterial invasion, leading to bacterial keratitis. A study from China showed that the prevalence of previous infectious keratitis and active infectious keratitis was 0.192%, and bacterial keratitis accounted for 39.06% [49]. Bacterial keratitis clinically presents with epithelial defects, stromal inflammation, and ulceration [50], potentially leading to permanent visual impairment or blindness [51].

The commonly reported rabbit keratitis models were established by directly injecting the strain into the corneal stroma [52,53,54,55]. Two specialized models warrant particular attention, one is soft contact lenses associated rabbit keratitis model, which aimed to explore the role of the collagen-binding adhesin in keratitis. Rhem et al. [56] marked the corneal epithelium of the right eye of each rabbit with a 9-mm trepanation and then performed the debridement within this area. Soft contact lenses that had been incubated in tryptic soy broth containing 10⁸ CFU/mL S. aureus for 24 h at 35 °C were then placed on the corneas to infect the rabbits. Another one is an isolated rabbit keratitis model established by Marino et al. [54], which injected the strains into the isolated corneal stroma. This model is reproducible and can be used as a mechanism-based alternative to in vivo animal testing. Compared with other reported disease models, the inoculation dose required of the keratitis model is lower, about 100 to 10⁴ CFUs. The information about rabbit keratitis models induced by S. aureus is summarized in Table 4.

The reported studies using rabbit models were mainly focusing on exploring the treatment of keratitis caused by S. aureus. Fluoroquinolones are suitable drugs, for example, moxifloxacin [57], which exhibit superior antibacterial activity than ciprofloxacin and levofloxacin in the late treatment of keratitis. Azithromycin [53] is also useful, due to its anti-inflammatory and antibacterial properties, as well as its ability to reduce the production of bacterial toxins. In addition to antibiotics, Las A [58], a protease secreted by pseudomonas aeruginosa, silver nanoparticles [59] and cationic antimicrobial peptide [60] all work a treat in the treatment of keratitis.

Several studies have explored the pathogenesis of the disease using rabbit models. In the study by Dajcs et al. [61], α-toxin is regarded as the main virulence factor in S. aureus-induced keratitis, while γ-toxin exhibited weaker virulence than α-toxin, and β-toxin played a less significant role. The strain lacking the α-toxin gene failed to cause epithelial erosion in rabbit keratitis model. The collagen-binding adhesin [56] produced by S. aureus also promoted the infection of cornea, eyes exposed to it are more likely to develop bacterial keratitis.

Table 4. The summary information of S. aureus-induced keratitis models in rabbits.

Author	Rabbit Species	Strain	Dose	Inoculation Mode
McCormick [52]	New Zealand White rabbit	S. aureus 83254	100 CFUs	Inoculation by corneal stroma injection
Ikemoto [53]	Japanese white rabbit	ATCC 25923	4.7 × 106 CFU/mL, 30 μL	Inoculation by corneal stroma injection
Marino [54]	Normal rabbit eyes	S. aureus-7786, 815, 74CCH	5 × 105 CFU/mL 0.05 mL	Inoculation by intrastromal injection
Sanders [55]	New Zealand White rabbit	MRSA 1131	100 CFUs	Inoculation by corneal stroma injection
Rhem [56]	New Zealand White rabbit	S. aureus	Not provided	Inoculation by applying specially treated contact lenses
Aghamollaei [60]	New Zealand White rabbit	MRSA	100 CFUs	Inoculation by injection to the cornea
Barequet [58]	New Zealand White rabbit	MSSA, MRSA	1000 organisms	Inoculation by intrastromal injection to the center of the cornea

Table 4. The summary information of S. aureus-induced keratitis models in rabbits.

Author	Rabbit Species	Strain	Dose	Inoculation Mode
McCormick [52]	New Zealand White rabbit	S. aureus 83254	100 CFUs	Inoculation by corneal stroma injection
Ikemoto [53]	Japanese white rabbit	ATCC 25923	4.7 × 106 CFU/mL, 30 μL	Inoculation by corneal stroma injection
Marino [54]	Normal rabbit eyes	S. aureus-7786, 815, 74CCH	5 × 105 CFU/mL 0.05 mL	Inoculation by intrastromal injection
Sanders [55]	New Zealand White rabbit	MRSA 1131	100 CFUs	Inoculation by corneal stroma injection
Rhem [56]	New Zealand White rabbit	S. aureus	Not provided	Inoculation by applying specially treated contact lenses
Aghamollaei [60]	New Zealand White rabbit	MRSA	100 CFUs	Inoculation by injection to the cornea
Barequet [58]	New Zealand White rabbit	MSSA, MRSA	1000 organisms	Inoculation by intrastromal injection to the center of the cornea

6. Rhinosinusitis

Chronic rhinosinusitis (CRS) is a prevalent chronic disease that significantly impairs patients’ quality of life and substantially increases healthcare costs [62,63]. CRS manifests in two primary clinical subtypes: nasal polyp and non-nasal polyp types, affecting approximately 5% to 15% of the population [64]. Biofilms exist on the mucosa of patients with CRS, which accounts for the difficulty in curing [65]. S. aureus is a prevalent biofilm-forming pathogen frequently isolated from patients with CRS [66].

To investigate the relationship between biofilms of S. aureus and the occurrence of CRS, Jia et al. [67] created a simple rabbit model of S. aureus biofilms-associated sinusitis by cutting the skin and periosteum on the dorsum of the rabbit nose and drilling into the maxillary sinus which was then inserted through the hole with a cylindrical absorbable gelatin sponge, and finally, a syringe was used to inoculate the bacterial suspension into the sinuses to infect rabbits. All model rabbits developed sinusitis confirmed by both CT and the presence of mucopurulent discharge in the maxillary sinus. The model closely resembled human rhinosinusitis. Wu et al. [68] utilized the same method with Jia to study the immune response to biofilms. The results showed that the expression of IL-1β, IL-8 and TNF-α increased while IL-4 and IL-5 decreased, which portended biofilm formation and extensive damage to epithelial cells.

Ciliary dysfunction is one of the causes of rhinosinusitis, the model of Min et al. [69] has shown that high concentrations of S. aureus SEA can inhibit ciliary motility. Long-term exposing to SEB [64] also leads to nasal mucosal thickening and inflammatory cell infiltration.

Owing to the limitations of antibiotic resistance and difficulty in administration in traditional topical treatment, rabbit models have been extensively utilized to explore alternative treatment approaches. Bleier et al. [70] presented a novel approach that antibiotics-impregnated chitosan glycerophosphate was implanted into the sinus, which can prolong the washout period of antibiotics and provide continuous local drug delivery. Corticosteroids [71] can serve as an adjunctive therapy to antibiotics for the treatment of sinusitis, accelerating the healing process. Jia et al. utilized 384 mg/L silk fibroin-nano silver solution [72] for local treatment and results showed that the biofilm was completely removed and the damaged epithelium was recovered.

New Zealand white rabbit was the primary species utilized in the rhinosinusitis model. Unlike the previously described models, Min et al. [69] directly induced the rabbit models by using the toxins of S. aureus. Table 5 summarizes the reported rabbit rhinosinusitis models, revealing that all models with traumatism blocked the sinuses. The inoculation dose of S. aureus ranged from 10⁶ to 10⁸ CFUs.

Table 5. The summary information of S. aureus-induced rhinosinusitis models in rabbits.

Author	Rabbit Species	Infectious Agents	Dose	Inoculation Mode
Karasen [73]	New Zealand White rabbit	inactivated S. aureus	1.1 × 109 0.2 mL	Inoculation by percutaneously applying to the maxillary sinus
Min [69]	New Zealand White rabbit	enterotoxin A (SEA) of S. aureus	0.3 ng/mL 2 mL 30 ng/mL 2 mL	Inoculation by percutaneously injecting
Uslu [74]	New Zealand White rabbit	S. aureus CMF-1	108 CFU/mL 0.2 mL	Inoculation by percutaneously injecting to maxillary sinus cavity
Sütbeyaz [71]	New Zealand albino rabbit	ATCC 25923	9 × 108 CFU/mL 0.5 mL	Inoculation by injecting to maxillary cavity
Dong [75]	New Zealand rabbit	ATCC 25923	1 × 108 CFU/mL	Inoculation by injecting to maxillary sinus
Bleier [70]	New Zealand White rabbit	S. aureus	4.0 × 108 CFUs	Inoculation by instilling into the sinus
Jia [67,72]	New Zealand White rabbit	S. aureus 006	1 × 106 CFU/mL 0.5 mL	Inoculation through the hole of maxillary sinus

Table 5. The summary information of S. aureus-induced rhinosinusitis models in rabbits.

Author	Rabbit Species	Infectious Agents	Dose	Inoculation Mode
Karasen [73]	New Zealand White rabbit	inactivated S. aureus	1.1 × 109 0.2 mL	Inoculation by percutaneously applying to the maxillary sinus
Min [69]	New Zealand White rabbit	enterotoxin A (SEA) of S. aureus	0.3 ng/mL 2 mL 30 ng/mL 2 mL	Inoculation by percutaneously injecting
Uslu [74]	New Zealand White rabbit	S. aureus CMF-1	108 CFU/mL 0.2 mL	Inoculation by percutaneously injecting to maxillary sinus cavity
Sütbeyaz [71]	New Zealand albino rabbit	ATCC 25923	9 × 108 CFU/mL 0.5 mL	Inoculation by injecting to maxillary cavity
Dong [75]	New Zealand rabbit	ATCC 25923	1 × 108 CFU/mL	Inoculation by injecting to maxillary sinus
Bleier [70]	New Zealand White rabbit	S. aureus	4.0 × 108 CFUs	Inoculation by instilling into the sinus
Jia [67,72]	New Zealand White rabbit	S. aureus 006	1 × 106 CFU/mL 0.5 mL	Inoculation through the hole of maxillary sinus

7. Meningitis

Meningitis is mainly classified into blood-borne and post-neurosurgical types, with hematogenous infections demonstrating significantly higher mortality rates [76]. S. aureus, the prevalent pathogen of bacterial meningitis [77], can cause both community-associated and hospital-associated meningitis, with the latter being the leading cause of death [78], particularly MRSA posing a challenge for the treatment of meningitis.

Reported rabbit meningitis models were established by inoculating S. aureus into the cisterna magna of rabbits. The New Zealand White rabbit was predominantly utilized in these experiments. Discrepancies occurred in the strains and dose of S. aureus, and the dose ranged from 10⁵ to 10⁸ CFUs. 25-gauge spinal needle and 22-gauge syringe were the main injection tools [78,79]. The summarized information of rabbit meningitis models is shown in Table 6.

The blood-brain barrier poses a significant challenge in the treatment of meningitis, and the reported rabbit meningitis model has made a great contribution to the treatment of meningitis. Linezolid has good permeability, and the concentration of it in human cerebrospinal fluid can reach 30% to 70% of the concentration in serum [80]. Calik et al. [81] found linezolid at a concentration of 20 mg/kg was equally effective as vancomycin, which is the first-line treatment for meningitis. Fusidic acid is a highly potent staphylococcal bacteriostatic agent with high permeability to the central nervous system; however, Ostergaard et al. [79] found that when it was used in combination with the fungicide methicillin, there would be a significant antagonistic effect. These findings suggest that antibiotics should be used rationally.

Table 6. The summary information of S. aureus-induced meningitis models in rabbits.

Author	Rabbit Species	Strain	MIC	Dose	Inoculation Mode
Ostergaard [79]	New Zealand White rabbit	S. aureus E2371	Fusidic acid 0.125 mg/L	1 × 107 CFUs	Inoculation by intracisternal injection
Sipahi [77]	New Zealand White rabbit	S. aureus ATCC 43300	Vancomycin 1 mg/L	1 × 107 CFU/mL 0.3 mL	Inoculation by injecting into the cisterna magna
Cabellos [82]	New Zealand White rabbit	MRSA-COL strain-GISA (Mu50, ATCC 700699)	Vancomycin 1 mg/L, Vancomycin 8 mg/L	1 × 108 CFU/mL	Inoculation by injecting into the cisterna magna
Gerber [83] Stucki [76]	New Zealand rabbit	MSSA 1112	Vancomycin 1 mg/L	1 × 105 CFUs	Inoculation by injecting into the subarachnoid space
Bardak-Ozcem [84] Calik [81]	New Zealand White rabbit	S. aureus ATCC-43300	Vancomycin 1 mg/L	1 × 106 CFU/mL 0.5 mL	Inoculation by injecting into the cisterna magna
Mermer [78]	New Zealand rabbit	S. aureus ATCC-43300	Vancomycin 1 mg/L	2 × 107 CFU/mL 0.3 mL	Inoculation by intracisternal injection

Table 6. The summary information of S. aureus-induced meningitis models in rabbits.

Author	Rabbit Species	Strain	MIC	Dose	Inoculation Mode
Ostergaard [79]	New Zealand White rabbit	S. aureus E2371	Fusidic acid 0.125 mg/L	1 × 107 CFUs	Inoculation by intracisternal injection
Sipahi [77]	New Zealand White rabbit	S. aureus ATCC 43300	Vancomycin 1 mg/L	1 × 107 CFU/mL 0.3 mL	Inoculation by injecting into the cisterna magna
Cabellos [82]	New Zealand White rabbit	MRSA-COL strain-GISA (Mu50, ATCC 700699)	Vancomycin 1 mg/L, Vancomycin 8 mg/L	1 × 108 CFU/mL	Inoculation by injecting into the cisterna magna
Gerber [83] Stucki [76]	New Zealand rabbit	MSSA 1112	Vancomycin 1 mg/L	1 × 105 CFUs	Inoculation by injecting into the subarachnoid space
Bardak-Ozcem [84] Calik [81]	New Zealand White rabbit	S. aureus ATCC-43300	Vancomycin 1 mg/L	1 × 106 CFU/mL 0.5 mL	Inoculation by injecting into the cisterna magna
Mermer [78]	New Zealand rabbit	S. aureus ATCC-43300	Vancomycin 1 mg/L	2 × 107 CFU/mL 0.3 mL	Inoculation by intracisternal injection

8. Endocarditis

Infective endocarditis (IE) is a serious intravascular infectious disease, in which the cardiac endothelium is involved, and IE is characterized by the formation of cauliflower-like vegetations on the heart valves [85]. The classification of IE is based on the location of infection, with four major categories: native valve endocarditis (NVE), prosthetic valve endocarditis (PVE), right-sided endocarditis, and device-related endocarditis [86]. S. aureus-induced IE presents high mortality and morbidity, especially caused by MRSA.

Rabbit models are commonly used for studying IE. The reported studies described two types of rabbit endocarditis models: One is a catheter-associated model in which the catheter was introduced into the left ventricle through the aortic valve or the right ventricle through the tricuspid valve, resulting in valve lesions and removal of the catheter, followed by intravenous inoculation of S. aureus to infect the rabbits, such as the models of Spaulding et al. [85] and Bastien et al. [87]. The other is intracardiac foreign bodies-associated model, the catheter was kept as a foreign body and held in place for the duration of the trial, and intravenously injected strains, like Huang et al. [88] and Chambers et al. [89]. Information of rabbit endocarditis models is summarized in Table 7. From the table, we can see that New Zealand white rabbit remain the most used species, and doses of S. aureus ranging from 10⁵ to 10⁸ CFUs. Except for the study of Wang et al. [90], the majority of experimental catheter locations were on the left side of the heart, causing left-sided endocarditis.

Vancomycin remains the first choice for the treatment of IE caused by MRSA [91]. Researchers have shown interest in using rabbit models to investigate alternative treatment strategies. The combination of vancomycin with other antibiotics, such as cloxacillin [91], produced a synergistic bactericidal effect against MRSA. Telavancin was effective against other glycopeptide-resistant MRSA strains [92] and was more active than vancomycin against the vancomycin-intermediate S. aureus [93]. The combination of daptomycin and β-lactam improved the survival of rabbits compared with the single drug treatment group. LSVT-1701 [88], a lyase encoded by bacteriophage, effectively reduces the number of MRSA in the experimental body when combined with daptomycin. Exebacase [94] is a direct cell wall cleaving agent that rapidly hydrolyzes the cell wall of S. aureus, and has a positive effect on the size, weight, and MRSA number of vegetations produced in IE when associated with daptomycin.

Table 7. The summary information of S. aureus-induced endocarditis models in rabbits.

Author	Rabbit Species	Strain	Dose	Inoculation Mode	Catheter Location and Duration
Huang [88]	Rabbit	MRSA MW2	2 × 105 CFUs	Inoculation by intravenously injecting	left ventricle 48 h
Spaulding [85]	New Zealand white rabbit	USA200, USA300, USA400, FRI1169, Newman, COL	Not provided	Inoculation by intravenously injecting after removing catheter	aortic valve 2 h
Chambers [89]	New Zealand white rabbit	S. aureus-76, HIP5836	106 CFUs	Inoculation by intravenously injecting	aortic valve 48 h
Madrigal [93]	New Zealand white rabbit	MRSA-COL, HIP 5836	106 CFUs	Inoculation by intravenously injecting	aortic valve 48 h
Bastien [87]	New Zealand white rabbit	S. aureus isolated from non-IE SAB patients	1–4 × 107 or 4.5–5.2 × 108 CFUs	Inoculation by ear marginal vein injection after the removal of the catheter	aortic valve 2 h
Chan [95]	New Zealand White rabbit	S. aureus COL	107–108 CFUs	Inoculation by intravenously injecting	left ventricle 48 h
Asseray [96]	New Zealand rabbit	MecA+ MecA−	108 CFUs	Inoculation by intravenously injecting	left ventricle 24 h
Shah [94]	New Zealand White rabbit	MRSA MW2	5 × 105 CFUs	Inoculation by intravenously injecting	left ventricle 48 h
Castañeda [91]	New Zealand White rabbit	MSSA-678, 277	105 CFU/mL 1 mL	Inoculation by intravenously injecting	left ventricle 24 h
Chambers [97]	New Zealand White rabbit	S. aureus CB5054	107 CFUs	Inoculation by ear marginal vein injection	left ventricle 48 h
Wang [90]	New Zealand White rabbit	ATCC 29213	8 × 107 CFUs	Inoculation by ear marginal vein injection	tricuspid valve

Table 7. The summary information of S. aureus-induced endocarditis models in rabbits.

Author	Rabbit Species	Strain	Dose	Inoculation Mode	Catheter Location and Duration
Huang [88]	Rabbit	MRSA MW2	2 × 105 CFUs	Inoculation by intravenously injecting	left ventricle 48 h
Spaulding [85]	New Zealand white rabbit	USA200, USA300, USA400, FRI1169, Newman, COL	Not provided	Inoculation by intravenously injecting after removing catheter	aortic valve 2 h
Chambers [89]	New Zealand white rabbit	S. aureus-76, HIP5836	106 CFUs	Inoculation by intravenously injecting	aortic valve 48 h
Madrigal [93]	New Zealand white rabbit	MRSA-COL, HIP 5836	106 CFUs	Inoculation by intravenously injecting	aortic valve 48 h
Bastien [87]	New Zealand white rabbit	S. aureus isolated from non-IE SAB patients	1–4 × 107 or 4.5–5.2 × 108 CFUs	Inoculation by ear marginal vein injection after the removal of the catheter	aortic valve 2 h
Chan [95]	New Zealand White rabbit	S. aureus COL	107–108 CFUs	Inoculation by intravenously injecting	left ventricle 48 h
Asseray [96]	New Zealand rabbit	MecA+ MecA−	108 CFUs	Inoculation by intravenously injecting	left ventricle 24 h
Shah [94]	New Zealand White rabbit	MRSA MW2	5 × 105 CFUs	Inoculation by intravenously injecting	left ventricle 48 h
Castañeda [91]	New Zealand White rabbit	MSSA-678, 277	105 CFU/mL 1 mL	Inoculation by intravenously injecting	left ventricle 24 h
Chambers [97]	New Zealand White rabbit	S. aureus CB5054	107 CFUs	Inoculation by ear marginal vein injection	left ventricle 48 h
Wang [90]	New Zealand White rabbit	ATCC 29213	8 × 107 CFUs	Inoculation by ear marginal vein injection	tricuspid valve

9. Conclusions

In this review, we have summarized the recent advancements of application of rabbit models for studying S. aureus-induced infectious diseases. The research principally encompasses two aspects: (1) the pathogenic mechanisms of S. aureus, including the cytotoxic effects of PVL in pneumonia and SSTIs, and the inhibitory effect of SEA on cilia in sinusitis; (2) the treatment of S. aureus infections, such as providing novel drug delivery approaches for osteomyelitis and sinusitis; for the treatment of drug-resistant strains, especially MRSA and VRSA, combination regimens such as “combination of vancomycin and cloxacillin” or other non-antibiotic drugs are provided.

Beyond the above diseases, rabbit models have also been widely used to address other clinically relevant S. aureus infections, such as surgical wound infections [98], implant-associated infections [99,100], and septic shock [101]. In these studies, rabbits possessed certain advantages as a model organism, including similar skin thickness [102,103] and anatomical structure [67] to humans, suitable body size and analogous sensitivity to superantigens and PVL [15,104], etc.

However, there remain areas where rabbit models can be further optimized, such as addressing issues like excessive inoculation dosage mentioned earlier and improving the accuracy of the rabbit endocarditis model [90]. Additionally, rabbits exhibit limited resistance to environmental stressors. Even minor disturbances, such as slight environmental noise, can trigger diarrhea [105]. In pharmacokinetic studies, rabbits have the limitation of higher compound consumption and higher cost compared to rodents. No single animal is suitable for all studies, and researchers should select the appropriate animal model according to the purpose of the study; for example, rabbits were chosen over rats for evaluating S. aureus superantigen activity due to rodents’ inherent resistance [106]; mice are preferred for transgenic research owing to their large pronuclei of zygotes and manipulable embryonic stem cells [107].

In conclusion, rabbits have been proven to be excellent animal models for studying S. aureus infections, with great progress having been made. It is anticipated that future rabbit models will exhibit manifestations that more closely resemble human infectious diseases caused by S. aureus and contribute to overcoming the existing challenges.