1Department of Medicine, Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, 2Geffen School of Medicine at UCLA
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Hady, W. A., Bayer, A. S., Xiong, Y. Q. Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat. J. Vis. Exp. (64), e3863, doi:10.3791/3863 (2012).
Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.
1. Preparing the MRSA Strains for Infection
2. Preparing Surgery Catheters
Cut polyethylene tubing (PE10; Becton Dickinson, order no. 427401) to 10 cm lengths, and melt one end by pressing the tip with a sterile forceps. The purpose of sealing one end of the catheter is to avoid bleeding during catheterization.
3. Pre-surgery Preparation and Anesthesia
4. Surgical Procedure
5. MRSA Infection
6. Sacrifice Rats and Culture Target Tissues
7. Representative Results
Immediately after inserting the catheter into the artery and pushing down the catheter about 4-5 cm toward the heart, a resistance will be experienced. If no resistance is sensed, the catheter may not have successfully been inserted into left side of the heart chamber, which may affect the placement of catheter. A correct catheter placement published previously9 is shown in Figure 1.
Part of MRSA infection samples need to be quantitative cultured the make sure the exact viable bacterial count for the infection and purity of infection sample. In addition, the organisms recovered from vegetations should be the same as those used in the inoculum.
Table 1 presents an example of virulence of a S. aureus strain in the rat endocarditis model which was published previously9. All animals challenged with inocula of 105 and 106 cfu and sacrificed between 3 to 6 days after infections developed endocardits with high S. aureus densities in cardiac vegetations, as well as kidneys and spleen (Table 1). Rats with sterile valve cultures are considered uninfected.
Figure 1. The catheter is in a correct place (left-side of heart chamber), and numerous vegetations are visible around the aortic valves9.
|Inoculum (no. of animas)||Mean log10 CFU/g of tissue ± SDa in:|
|106 CFU/animal (9)||10.36 ± 0.85||7.30 ± 0.64||6.70 ± 0.57|
|105 CFU/animal (8)||9.93 ± 0.53||7.14 ± 0.53||6.44 ± 0.63|
|104 CFU/animal (7)||3.46 ± 0.50*||1.81 ± 0.74*||1.58 ± 0.59*|
a SD, standard deviation. P < 0.001 with respect to 105 or 106 CFU-challenged animals.
Table 1. S. aureus density in cardiac vegetations with different inocula in the rat endocarditis model9.
The rat endocarditis is an important and well-characterized animal model for in vivo studies in the pathogenesis and antimicrobial agents in the treatment of bacterial infections 9-11. In addition, rat endocarditis model represents a composite of acute and sub-acute infections, and closely mimics human counterpart and human native valve endocarditis. Moreover, accompanied by an in dwelling catheter, it represents a classic biofilm-associated infection, a common and difficult issue in clinical settings 12. Most importantly, results generated with the rat endocarditis model are highly reproducible.
The pathogenicity of S. aureus in the rat endocarditis model, as other animal models, may vary depending on a number of parameters, including inoculum dose, bacterial growth phase (log vs. stationary), age of animals, and animal genetic background 9, 13. Surgical success rates are highly dependent upon the technical expertise and surgical skills of the researcher. Success in the surgery can be measured by: 1) the number of animals that recover from the surgery; 2) catheter in a correct place at time of sacrifice; and 3) vegetations present on cardiac valves.
In summary, the rat endocarditis is a practicable, reproducible and important model that mimics the clinical situation closely. Therefore, it is appropriate for all fields of sepsis research, including microbial pathogenesis and efficacy of antimicrobial agents in the treatment of experimental endocarditis syndromes.
No conflicts of interest declared.
This work was supported by the U.S. National Institutes of Health [grant R01AI-39108 to A.S.B.] and the American Heart Association [grants SDG 0630219N and AID 09GRNT2180065 to Y.Q.X.].
|Blood agar plate, 5% sheep blood in Trypticase Soy agar(TSA)||Hardy Diagnostics||A10BX|
|Trypticase Soy broth (TSB)||BD Biosciences||211825|
|Shaking incubator||Labnet International||I5311-DS|
|Polyethylene tubing||BD Biosciences||427401|
|Catheter introducer||BD Biosciences||6999|
|Isofluorance||Western Medical Suppy, Inc||2147||Follow safety and handling information|
|Surgical Instruments||Fine Science Tools and Biomedical Research Instruments, Inc.||Find the instruments from the two companies for your needs|