A-1320
GRIPE
Successful Growth of Staphylococcus epidermidis in the Neutropenic Mouse Thigh Infection Model (NMTIM) without the Use of a Foreign Body 1
1
1
1
1
Luz M. Leiva , Sharon Imbett , Jean P. Gómez , Mateo González , Carlos A. Rodriguez , Maria Agudelo
1,3
, Javier M. González
1,2
and Omar Vesga
1,3
Omar Vesga, MD
.
Director GRIPE and Professor of Medicine
1. University of Antioquia Medical School, Medellin, Colombia. Financed by FCRVM and UdeA.
Section of Infectious Diseases Departments of Internal Medicine and Pharmacology
2. Clínica Cardiovascular Congregación Mariana, Medellín, Colombia.
University of Antioquia Medical School
3. Hospital Universitario San Vicente Fundación, Medellín, Colombia
Calle 62 No. 52-5], Lab 630, Medellín, Colombia.
[email protected]
ABSTRACT
RESULTS
Background: Staphylococcus epidermidis is one of the most important causes of infections associated with medical devices like catheters and prosthesis. All published animal models require a foreign body for a successful infection otherwise, bacterial growth is minimal or even negative. We aimed to develop a model with active growth, appropriate for pharmacodynamics studies, but without the need of a foreign body. Methods: neutropenic MPF Udea:ICR (CD-2) female mice were inoculated in each thigh with 0.1 mL of a log-phased culture containing 5 log10CFU/mL. We selected a clinical strain of methicillin-resistant S. epidermidis (MRSE) that does not produce biofilm (Christensen’s tube method) and compared bacterial growth in the NMTIM without and with the addition of 5% porcine mucin to the inoculum. Animals were sacrificed immediately after inoculation, 2 hours later (2 mice at each point), and at 26 hours (3 mice) and bacterial counts determined by plating serial dilutions of the thighs’ homogenates (CFU/g). Independent repeats of the mucin model demonstrated its reproducibility. In vivo growth of S. epidermidis GRP-0150 Results: Experiment Without mucin With mucin 1 With mucin 2 Mean (SEM) Mean (SEM) Mean (SEM) Inoculum 4.]6 5.04 5.13 Time -2h 4.33 (0.13) 4.22 (0.04) 4.21 (0.01) Time 0h 4.7] (0.02) 4.75 (0.02) 5.17 (0.00) Time 24 2.52 (0.2]) 8.78 (0.05) ].35 (0.07) Net growth
-2.27
4.03
Discussion
In vivo growth standardization of S. epidermidis GRP-0150 in the NMTIM with and without mucin is shown in Fig. 1 and the data are detailed in Table 1. The model with mucin was repeated independently to assess reproducibility. Two hours after infection (0h), mice in both groups had the same bacterial load, but 24 hours later S. epidermidis had decreased in the control group (-2.27 log10 CFU/g) while it was able to grow more than 4 logs in the mucin group reaching ~] log10 CFU/g in 24 hours with minimal variance. The successful mucin model was employed to compare the PD of vancomycin against S. epidermidis and S. aureus. The PD profile of vancomycin (VAN) is shown in Figure 2, and PK/PD parameters are shown in the Table 2. PD parameters (PD) are compared with the S. aureus GRP-0057.
4.18
Mucins are heavily glycosylated proteins produced by epithelial cells and a potential source of amino acids and sugars to increase PIA or another adhesion factors. Muscle cells do not produce mucin, explaining the absence of adherence factors in the thigh infection model and the negative growth after infection with S. epidermidis (Fig. 2). The addition of mucin to the inoculum enabled the replication of S. epidermidis in the NMTIM without the use of a foreign body.
Figure 1
Conclusions: mucin addition to the infecting inoculum of a biofilm negative strain of MRSE is associated with high and reproducible bacter ial growth in the NMTIM with very small variance. This model is useful to study antimicrobial pharmacodynamics in the absence of a foreign-body.
Table 1 In vivo growth of S. epidermidis GRP-0150 without (control group) and with mucin in the NMTIM
In v iv o g r o w t h o f S . e p id e r m id is G R P -0 1 5 0
1 0
introduction
1 0
W ith o u t m u c in W ith m u c in E x p .1
9
Initial adhesion to the polymer surface is necessary to obtain S. epidermidis infection. This process and biofilm formation are mediated by nonspecific forces such as surface charge, specific adhesins and a polysaccharide adhesion (PSA) protein in the early phases. The later phases, in which organisms adhere to one another and elaborate biofilm, are mediated by polysaccharide intracellular adhesion (PIA) proteins whose expression depend on the availability of glucose and are required to produce abscesses in experimental animal infections []].
9
The antibiotic effects on growing and not growing bacteria are different, and actively reproducing organisms are needed to determine the magnitudes of the PDP required to treat infections like bacteremia. Compared with the professional pathogen S. aureus, the saprophytic but common S. epidermidis is eliminated with 2.5-3.0x lower vancomycin exposure. It helps to understand in vitro PD data and supports the different clinical break-points for S. epidermidis.
Bacterial adherence is critical in the pathogenesis of staphylococcal infections, particularly in biomaterial-based infections [4]. The capacity of S. epidermidis to produce slime is important in the model of foreign body infection [5]. Animal models of S. epidermidis infections have demonstrated the requirement of large bacterial inocula and either an unusual bacterial strain or a foreign body to cause infection otherwise, bacterial growth is minimal or even negative.
10
Log
Coagulase-negative staphylococci (CoNS) and, Staphylococcus epidermidis in particular, are ubiquitous members of the skin and mucosal human microflora that commonly cause infections [1]. For instance, S. epidermidis is the most common cause of central line-associated bloodstream infections (CLABSIs) and the second most common cause of surgical site infections (SSIs) [2,3]. Additionally, S. epidermidis is the principal organism responsible for infections related to prosthetic medical devices.
C F U /g
W ith m u c in E x p .2
8
8
7
7
6
6
5
5
4
4
3
3
2
2
-2
Mucin has shown promise to facilitate in vivo growth of different gram-positive bacterial species, as has been the case of Streptococcus pneumoniae and Enterococcus spp. in murine models of infection [6,7]. Here, we aimed to develop a NMTIM with S. epidermidis appropriate to study antimicrobial pharmacodynamics without the use of a foreign body, and tested the model comparing the PD of vancomycin against S. epidermidis and S. aureus.
0
CONCLUSIONS
2 4
The addition of mucin to the infecting inoculum of a biofilm-negative strain of MRSE is associated with high and re-
T im e (h )
producible bacterial growth in the NMTIM with minimal variance. This model is useful to study antimicrobial pharmacodynamics against this organism in the absence of a foreign-body.
Figure 2
METHODS
Table 2
V A N v s . S . a u r e u s G R P - 0 0 5 7 a n d S . e p id e r m id is G R P - 0 1 5 0
Strains, inocula preparations and experimental groups. Staphylococcus epidermidis GRP-0150, a meticillin-resistant clinical strain that does not produce biofilm (evaluated by Christensen´s tube
2
method) was used. The vancomycin MIC was 4 mg/L. It was stored at -70˚C (Glacier NU]668, Nuaire®, Plymouth, MN, USA) and resuscitated in Brain Heart Infusion agar (Becton Dickinson®). Re-
S . a u re u s
1
covered bacteria were grown at 37˚C in two successive steps in Trypticase Soy Broth (Becton Dickinson®) under aerobic atmosphere until reaching an OD580=0.358, which corresponds to a bacteri-
S . e p id e r m id is
8
group, we added porcine mucin to a 5% final concentration (Sigma-Aldrich®, Saint Louis, MO, USA). Control mice received the same inoculum but without mucin. Study animals. Six-week-old MPF Udea:ICR(CD-2) female mice weighting 23-27 g were rendered neutropenic (
