Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Clinical Science
Non Technical Summary
Mares with a chronic bacterial infection in their uterus that is resistant to traditional antibiotic therapy may have reduced fertility. Bacteria involved in chronic uterine infections may be capable of forming a biofilm, a sugar-like matrix consisting of biological polymers that coats bacterial colonies adherent to the wall of the uterus in a 'protective blanket'. The biofilm matrix is able to provide protection from the natural uterine immune system by altering the movement and function of white blood cells, and preventing antibodies from binding to bacteria. In addition, biofilms confer tolerance to antibiotic therapy by reducing the amount of drug that reaches the bacteria and altering metabolism and growth of the bacteria so that antibiotics are not as effective. Ultimately, biofilms are associated with a population of bacteria that are protected and tolerant to antimicrobial agents are able to propagate and further disperse to other infection sites. Preliminary data from our lab confirms that biofilm infections can be established in the uterus of the mare using an in vivo model. In addition, we have recently determined in laboratory studies that the best efficacy in killing the bacteria and disrupting the biofilm matrix is with a combination of antibiotics and non-antibiotic compounds. However, it is unknown if the proposed combination therapy will have a similar effect at eliminating a biofilm associated infection when treatment is performed in the horse. The goals of this proposal are to 1) determine if the combination of antibiotics and non-antibiotics are capable of reducing bacterial viability and biofilm matrix utilizing a model of biofilm associated infectious endometritis, and 2) evaluate the mare's immune response during and after treatment.
Animal Health Component
90%
Research Effort Categories
Basic
10%
Applied
90%
Developmental
(N/A)
Goals / Objectives
Hypothesis 1: Treatment of an established P. aeruginosa biofilm with a combination of ceftiofur plus Tris-EDTA will result in elimination of biofilm material and viable bacteria as compared to untreated control or ceftiofur and Tris-EDTA independently. Specific Aim 1: Determine the viability of P. aeruginosa and detect biofilm components in the uterine lumen following treatment of an established P. aeruginosa biofilm infection with 1) ceftiofur, 2) Tris-EDTA, 3) ceftiofur plus Tris-EDTA and 4) untreated control A model of in vivo bacterial endometritis will be used to establish a P. aeruginosa infection involving a preformed biofilm. Following development of the biofilm-associated infection, mares will be randomly assigned to the following treatments 1) ceftiofur (n=5), 2) Tris-EDTA (n=5), 3) Ceftiofur plus Tris-EDTA (n=5) and 4) untreated control (n=5). Following treatment, the uterus will be assessed for viable bacteria (bacterial culture), presence of adherent material on the endometrial surface (hysteroscopy with guided biopsy sampling) and confirmation of the adherent material as a bacterial biofilm (immunohistochemistry). The goal is to determine if treatment was able to kill the bacteria and eliminate the biofilm material on the surface of the endometrium.Hypothesis 2: Treatment with ceftiofur plus Tris-EDTA will result in a reduction in inflammatory cells within the uterine lumen. Specific Aim 2: Monitor the inflammatory response (in the uterine lumen and endometrium) prior to bacterial inoculation, after establishment of a P. aeruginosa biofilm, and after treatment with 1) ceftiofur, 2) Tris-EDTA, 3) ceftiofur plus Tris-EDTA and 4) untreated control. During sample collection for Specific Aim #1 endometrial cytology samples will be collected and evaluated by a non-biased evaluator to determine the level of inflammatory cells present in the uterine lumen. Additionally, biopsy samples collected from Specific Aim #1 from areas with and without adherent material will be evaluated by a non-biased pathologist to monitor the degree of inflammatory cell infiltrate into the endometrium. The goal is to monitor inflammation in the uterine lumen and endometrium prior to inoculation and after treatment of a preformed biofilm.
Project Methods
Before enrolling in the study, mares (n=20) will have been evaluated and required to have a negative endometrial culture for aerobic growth, no detection of inflammatory cells on endometrial cytology, and no inflammatory cells on biopsy. This biopsy sample will also serve as a control sample for evaluation by IHC (presence of bacteria and exopolysaccharide Pel).Mares will be synchronized and allowed to ovulate so that all horses are at the same stage of the estrous cycle. Once ovulation is detected mares will be started on daily intramuscular injections of 200 mg of short acting progesterone for 5 days. On day 5 mares will be inoculated with 2 ml of a mixture of 3 different clinical isolates of P. aeruginosa at a concentration of 106 colony forming units/ml. The use of 3 strains of P. aeruginosa in the inoculation media reduces the risk of utilizing bacteria in the study with minimal ability to replicate or produce a biofilm in vivo. This model of infection has been utilized in our lab to readily establish a biofilm associated infection in 100% of the studies performed to date (9 of 9 mares) 8.The mares will continue to receive daily injections of short acting progesterone as the infection develops for 5 days at which point mares will be assigned in a non-biased manner to one of the following treatments: 1) ceftiofur (n=5), 2) Tris-EDTA (n=5), 3) ceftiofur plus Tris-EDTA (n=5) and 4) untreated control (n=5). Progesterone supplementation will be discontinued once treatment is initiated. To confirm the presence of the infection just prior to the first treatment a uterine culture and cytology will be performed; evidence of a uterine infection will include detection of Pseudomonas aeruginosa and inflammatory cells on cytology. Five mares per treatment group were selected as a power calculation indicated that if 4 of the 5 mares had similar outcomes a significant difference could be detected between groups.TreatmentsTreatment 1: A 60 ml uterine infusion containing 1 gram of ceftiofur sodium (16 mg/ml), Treatment 2: A 60 ml uterine infusion containing and 50 mM Tris and 3.5 mM EDTA, Treatment 3: A 60 ml uterine infusion containing 1 gram of ceftiofur sodium (16 mg/ml) and 50 mM Tris and 3.5 mM EDTA, Treatment 4: untreated control with no intrauterine treatments performed. Each treatment will be infused every 24 hours for a total of 3 intrauterine infusions. The uterine infusions will be performed using a sterile infusion pipette connected to a 60 ml syringe passed through the cervix of the mare and the treatment infused into the uterine lumen. The drugs, concentration and treatment period were selected based on common use in clinical practice 27.The untreated control mares and any non-effective treatments at eliminating the pre-formed biofilm as determined in Specific Aim 1 and 2 will be treated at the conclusion of the project with the most effective treatment as identified in Specific Aim 1 and 2.Specific Aim 1: Determine if viable P. aeruginosa and biofilm components are present in the uterine lumen following treatment with ceftiofur and/or Tris-EDTA. The goal is to evaluate the uterus 72 hours after the last treatment, in which residual ceftiofur and Tris-EDTA should no longer be present to alter the ability to cultivate organisms. This time period is selected based off reported intervals between antibiotic therapy and collection of a culture in horses with respiratory infections. All samples for Specific Aim 1 and 2 will be collected 72 hours after the last treatment.Aerobic CultureA sample for aerobic culture will be collected with a double guarded culture swab, the swab will be passed through the cervix of the mare and gently rolled against the endometrial surface for 15 seconds. The swab will then be placed in Amie's liquid media and cultured for growth of aerobic bacteria. Bacterial colonies detected will be submitted for identification using MALDI-TOF. The goal is to determine if viable bacteria are present in the uterine lumen following uterine treatment.HysteroscopyA hysteroscopy of the uterus will be performed using a sterilized 1.8 meter flexible endoscope. The endoscope will be guided through the cervix into the uterine lumen, the uterine lumen will be dilated with filtered air to allow visualization of the endometrial surface. The endometrial surface will be evaluated for the presence or absence of tissue adherent biofilm. Areas of the endometrium with adherent material will have a double guarded culture swab collected from the area and submitted for aerobic culture. A guided biopsy samples will be collected with tissue adherent biofilm and an adjacent area of the endometrium without adherent material. If no adherent material is detected a biopsy will be collected from the base of one uterine horn.The biopsy samples will be fixed Bouin's solution for 12-18 hours, followed by 70% ethanol in preparation for paraffin embedding. Paraffin embedded tissue will be sectioned from the tissue block and were prepared for IHC with a primary anti-Pseudomonas antibody and a labeled lectin that binds to the biofilm exopolysaccharide Pel. This method was used to evaluate tissue adherent biofilm on the endometrial surface in our laboratory 42.The goal of the hysteroscopy and analysis of biopsy samples is to determine if tissue adherent biofilm is present on the surface of the endometrium and if this adherent material contains viable bacteria and the bacterial specific components consistent with a biofilm. Successful treatment will result in an inability to detect viable bacteria or tissue adherent biofilm (grossly on hysteroscopy or microscopically by IHC) in the uterine lumen.Specific Aim 2: Monitor the inflammatory response following treatment (ceftiofur and/or Tris-EDTA) in the uterine lumen and endometrium following establishment of a P. aeruginosa infection involving a biofilm. Endometrial CytologyAt the same time as the samples are collected for aerobic culture (Specific Aim 1) an endometrial cytology sample will be collected. A double guarded cytology brush will be gently rolled in contact with the endometrium for 15 seconds, followed by the brush being gently rolled onto a glass slide, allowed to air dry, and stained with a modified Wright's stain. Samples will be evaluated by non-biased evaluator for the presence, quantity, and characterization of any inflammatory cells. The goal is to characterize the inflammatory response in the uterine lumen between treatment and the untreated control. Successful treatment will have a decrease in white blood cells as compared to the untreated control.Endometrial BiopsyThe biopsy samples embedded in paraffin blocks collected in Specific Aim 1 will be sectioned and stained with hematoxylin and eosin (H&E) stain. This slide will be evaluated by a non-biased pathologist for the degree and population of inflammatory cells in the endometrium. The goal is to compare the inflammatory response within the uterine tissue between treatments and the untreated control. Successful treatment will be associated with a decrease in white blood cells in the endometrium, and lack of adherent biofilm on the luminal surface.Data AnalysisIndividual comparisons will be made utilizing a simple t test. Categorical data will be analyzed via Chi-Square analysis. Treatment interactions and inflammation in the endometrium will be evaluated with a mixed model analysis of variance. Fixed variables will be collection period (prior to inoculation or post treatment) or treatment (between treatments and untreated control). Both main and interaction effects will be assessed, where the horse is considered a random effect. A p value of <0.05 will be considered significant.