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Practical Mouse Model to Investigate Therapeutics for Staphylococcus aureus Contaminated Surgical Mesh Implants

Open AccessPublished:November 23, 2022DOI:https://doi.org/10.1016/j.jss.2022.10.093

      Abstract

      Introduction

      The use of prosthetic mesh in hernia repair provides a powerful tool to increase repair longevity, decrease recurrence rates, and facilitate complex abdominal wall reconstruction. Overall infection rates with mesh are low, but for those affected there is high morbidity and economic cost. The availability of a practicable small animal model would be advantageous for the preclinical testing of prophylactics, therapeutics, and new biomaterials. To this end, we have developed a novel mouse model for implantation of methicillin-resistant Staphylococcus aureus–infected surgical mesh and provide results from antibiotic and immunotherapeutic testing.

      Materials and Methods

      Implantation of surgical mesh between fascial planes of the mouse hind limb was used to approximate hernia repair in humans. Surgical mesh was inoculated with methicillin-resistant Staphylococcus aureus to test the efficacy of antibiotic therapy with daptomycin and/or immunotherapy to induce macrophage phagocytosis using antibody blockade of the CD47 “don't eat me” molecule. Clinical outcomes were assessed by daily ambulation scores of the animals and by enumeration of mesh-associated bacteria at predetermined end points.

      Results

      A single prophylactic treatment with daptomycin at the time of surgery led to improved ambulation scores and undetectable levels of bacteria in seven of eight mice by 21 days postinfection. Anti-CD47, an activator of macrophage phagocytosis, was ineffective when administered alone or in combination with daptomycin treatment. Ten days of daily antibiotic therapy begun 3 days after infection was ineffective at clearing infection.

      Conclusions

      This fast and simple model allows rapid in vivo testing of novel antimicrobials and immunomodulators to treat surgical implant infections.

      Keywords

      Introduction

      Over the last 60 years, the implantation of prosthetic mesh has become commonplace in hernia repair surgery and has resulted in superior longevity as compared to tissue repairs.
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      Unfortunately, mesh hernia repairs are associated with a number of complications, the most feared of which is infection. The incidence of mesh infection depends on the anatomic location and the resultant tissue vascularity and the surgical approach. In general, mesh infections in inguinal hernia repair are low, especially when using minimally invasive techniques. These rates increase with open technique and with ventral abdominal wall repair and are reported as high as 6%-10% for open incisional hernia repair.
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      Once surgical mesh is infected, antibiotics are generally not sufficient to eradicate the infection and surgical explantation is required.
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      Mesh-related infections after hernia repair surgery.
      Secondary surgical repair then often becomes necessary but with an increased risk of failure and hernia recurrence. Thus, such infections are highly morbid to the patient and costly to the healthcare system.
      Methicillin-resistant Staphylococcus aureus (MRSA) is a common causative agent of both skin and soft tissue infections and implantable device-related infections. Chen et al. demonstrated that more than 50% of infected mesh recovered from abdominal wall repair sites was caused by S aureus.
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      Moreover, 63% of mesh infections following incisional herniorrhaphy were caused by MRSA contamination.
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      Animal models for hernia repair have been developed in rats, rabbits, pigs, and some mice,
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      but there has been surprisingly limited description of research focused on infected implants. Most of the literature describing small animal models of mesh implants involves the use of rats (53%), and of these studies, many undergo true hernia defect creation with subsequent mesh placement.
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      It would be much more expedient to use a mouse model and avail the powerful genetic tools available in mice to advance research in this area. However, the thickness of the abdominal wall in a mouse is inadequate to represent the tissue planes in a human. In the current report, we describe a cost-effective, simplified murine model using alternative mesh placement into the mouse thigh to study the treatment of contaminated surgical mesh in mice. Specifically, we examined the effect of antibiotic therapy with daptomycin and also immunotherapy with anti-CD47, previously shown to potentiate macrophage-mediated phagocytosis,
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      The role of CD47-SIRPα immune checkpoint in tumor immune evasion and innate immunotherapy.
      which is important in the clearance of numerous bacterial infections. Although daptomycin is not commonly used in surgical prophylaxis, we chose it for the development of the model because it is known to be effective against MRSA in mice
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      • et al.
      In vivo efficacy of daptomycin against methicillin-resistant Staphylococcus aureus in a mouse model of hematogenous pulmonary infection.
      and is also highly efficacious in treating patients with surgical site MRSA infections.
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      Daptomycin-rifampin for a recurrent MRSA joint infection unresponsive to vancomycin-based therapy.
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      Daptomycin for the treatment of surgical site infections.
      In addition, it is included in the evidence-based guidelines for the treatment of patients with MRSA infections.
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      Clinical practice guidelines by the Infectious Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children.

      Materials and Methods

      Ethics statement

      All animal studies and procedures were approved by the Institutional Animal Care and Use Committee at Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH). Work was carried out in accordance with the institutional guidelines for animal use and followed the guidelines and basic principles in the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals, the Animal Welfare Act, and the Guide for the Care and Use of Laboratory Animals and conformed to the guidelines of the NIH.

      Bacterial strains and inoculum preparation

      S aureus USA300 strain Los Angeles County clone was used in this study.
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      • et al.
      Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils.
      Bacteria were cultured in tryptic soy broth (TSB, Millipore) at 37°C with shaking at 225 rpm. Overnight culture was diluted 1:200 into fresh medium and cultured to the mid-logarithmic phase of growth (OD600 = 0.75). Subsequently, bacteria were collected by centrifugation (4°C; 4000 × g for 8 min), washed, and suspended in sterile, injection grade saline to the desired concentration (107 colony forming unit [CFU]/injection). The bacterial inoculum was kept on ice until inoculation and the concentration verified by plating serial 10-fold dilutions on tryptic soy agar plates (Millipore). CFUs were enumerated after 24 h of growth at 37°C.

      Mice

      Female (C57BL/10 x A.BY) F1 mice (Y10) were produced at Rocky Mountain Laboratories in an Association for Assessment and Accreditation of Laboratory Animal Care International approved facility. Parental mice were obtained from the Jackson Laboratory. Age-matched and gender-matched females between 8 and 16 weeks of age were used. The Y10 mouse strain used in this study has a fully functional SLC11a1 gene (formally known as Nramp1) that permits assessment of the role of macrophages during S aureus infection.
      • Blackwell J.M.
      • Goswami T.
      • Evans C.A.
      • et al.
      SLC11A1 (formerly NRAMP1) and disease resistance.
      ,
      • Vidal S.
      • Tremblay M.L.
      • Govoni G.
      • et al.
      The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene.
      It should be noted that pilot experiments for this study showed that the C57BL/6 strain of mice was extremely susceptible to MRSA infections with two log higher bacterial counts at day 6 than (C57BL/10 x A.BY) F1 mice (data not shown). C57BL/6 and C57BL/10 mice carry a known recessive mutation of the SLC11a1 gene that has pleiotropic effects on macrophage function, which results in high susceptibility to infections by Salmonella sp. and Mycobacterium bovis.
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      • Goswami T.
      • Evans C.A.
      • et al.
      SLC11A1 (formerly NRAMP1) and disease resistance.
      ,
      • Vidal S.
      • Tremblay M.L.
      • Govoni G.
      • et al.
      The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene.
      Because we were interested in studying CD47 blockade to enhance macrophage function, and because macrophages are important in bacteria clearance, the selection of a mouse strain with defective macrophage function was not appropriate and we chose to use (C57BL/10 x A.BY) F1 mice to obtain a dominant wild type SLC11a1 from the A.BY strain.

      Surgical preparation

      To prepare mice for surgery, each mouse was anesthetized with isoflurane and positioned in right lateral recumbency for shaving. The lateral surface of the left hind leg from below the knee to above the pelvis was shaved using a size 40 blade (Fig. 1A). When possible, shaving was completed 1 day prior to surgery to allow mice to naturally groom away small free hairs from the clipped area. On the day of surgery, mice were weighed and an intraperitoneal injection of ketamine (∼96 mg/kg) and xylazine (∼15 mg/kg) was administered to induce general anesthesia. Once each mouse was anesthetized, an ocular lubricant (Optixcare, CLC Medica) was applied to each eye and the surgical site was scrubbed with a 4% chlorhexidine gluconate surgical scrub (E-Z scrub, BD). Each mouse was then positioned on a sterile platform in right lateral recumbency and placed in front of the surgeons.
      Figure thumbnail gr1
      Fig. 1Implantation and infection of surgical mesh. (A) Overview photograph of a mouse in right lateral recumbency providing reference for the surgical approach. The solid black line is the approximate location of the femur and the dashed line depicts the incision site. (B) Incised skin has been retracted to expose the fascial surfaces of the musculature. The visible white band of tissue indicates the approximate separation of the quadriceps and hamstring muscles. The dashed line to the right of the white band indicates the location of blunt dissection to create the pocket depicted by the yellow arrow in (C). (D) The blue arrow indicates the mesh being inserted into the pocket. (E) The mesh (blue arrow) was positioned caudal to the femur and cranial to the sciatic nerve (indicated by the yellow arrow). (F) Instillation of USA300 S aureus inoculum deep into the pocket directly onto the mesh (black arrow) using micropipette and sterile tip. (G) and (H) show closure of the subcutaneous tissues with suture and skin closure with 9-mm staples. The mouse shown in these images was euthanized prior to manipulation for photographs.

      Mesh implantation and MRSA instillation

      Surgeons wore gowns, head covers, masks, sterile gloves, and used sterile instruments and “tips-only” technique. A 1-cm skin incision was made parallel to and slightly caudal to the femur (Fig. 1A, dotted line). The skin was retracted away from the incision to visualize the tissue. Caudal to the femur, a combination of adipose and connective tissue delineated the separation between biceps femoris and the vastus lateralis muscles (Fig. 1B, dotted line). Blunt dissection was used to create a small pocket between these muscles (Fig. 1C) and a ∼1.0 × 0.5 cm section of sterile Bard Mesh Flat Mesh (BD) with fire-polished edges was inserted parallel to the femur (Fig. 1D). Fire-polishing the cut mesh edges helped prevent the mesh from catching on tissue during the implantation and aimed to reduce discomfort from rough surfaces postimplantation. For experimental contamination of the mesh, a titrated MRSA suspension containing 107 CFU in 20 μL of sterile saline was applied directly onto the mesh surface within the surgical pocket (Fig. 1F). Pilot studies showed this dose to consistently cause unresolved chronic infection in untreated mice (data not shown). Controls included mice that received mock infection and S aureus–infected mice that were not treated. The fascia was approximated with a single cruciate suture (5-0 PDS II, Ethicon) (Fig. 1G) and the skin was closed with 9-mm wound clips (MikRon Precision, Inc.) (Fig. 1H). Each mouse was administered ∼ 1 mg/kg Buprenorphine-SR (Zoopharm) subcutaneously for pain management. A 1-mg/kg intraperitoneal injection of atipamezole (Zoetis) was administered to reverse the xylazine component of the anesthetic injection. Mice were recovered in their cages on a 42°C thermal plate for several hours and were observed daily following implantation and infection.
      Two surgeons were used to separate the procedure into “MRSA clean” and “MRSA dirty” sections. The first surgeon made the approach and placed the mesh and the second surgeon instilled the MRSA and closed the fascia and skin. Uninfected control mice were implanted first on each surgery day.

      Antimicrobial drug therapies

      Designated groups of animals were administered 250 μg daptomycin (Northstar Rx) in 100 μL via intraperitoneal injection (i.p.) immediately after surgery or beginning 3 days postinfection (dpi). Other groups received either 100 μg of anti-CD47 (BioXcell #BE0283) in 100 μL i.p. alone or in combination with 250 μg daptomycin. Treatment was administered as a single dose or daily for 10 consecutive days.

      Ambulation scoring

      All mice were monitored daily and scored in a randomized, blinded, manner for ambulation. Scoring criteria are as follows: 0: no changes in gait, 1: slight limp, 2: intermittent weight bearing, and 3: nonweight bearing. Mice were euthanized when they met end point criteria or by their scheduled euthanasia day postsurgery.

      Evaluation of bacteria attachment to the surgical mesh implants

      Upon removal, the surgical mesh was rinsed by careful submersion in 10 mL of sterile phosphate buffered saline (Sigma–Aldrich) to remove unattached bacteria. Mesh was subsequently placed in a tube containing 2 mL of sterile phosphate buffered saline. To free attached bacteria, samples were sonicated as described elsewhere.
      • Belyansky I.
      • Tsirline V.B.
      • Montero P.N.
      • et al.
      Lysostaphin-coated mesh prevents staphylococcal infection and significantly improves survival in a contaminated surgical field.
      Briefly, samples containing surgical mesh were placed in the ice water bath sonicator and sonicated at a frequency of 40 kHz for 5 min (Branson 2200, Branson Ultrasonics) followed by 10 min vortex. Samples were immediately diluted and 100 μL of serial 10-fold dilutions were plated on tryptic soy agar plates. Colonies were enumerated the next day after 24 h incubation at 37°C. For several experiments, bacteria were additionally plated on CHROMID MRSA plates (bioMerieux) to ensure that there was no contamination and recovered bacteria were MRSA.

      Histology and immunohistochemical staining

      A tissue sample from the upper leg was collected to evaluate tissue pathology due to mesh implantation and MRSA infection. After animal euthanasia, the skin was removed from the entire upper left leg and the region from the femoral head to the knee was removed as a single piece and placed in 10% neutral buffered formalin for 3 to 5 days. Following fixation, samples were processed by dehydration and embedding in paraffin. Sections were cut using a standard Leica microtome, placed on positively charged glass slides, and air-dried overnight at room temperature. On the following day, slides were heated in an oven at 60°C for 20 min. Pathology was assessed on hematoxylin and eosin (H&E)–stained sections. H&E staining was performed as per the manufacturer's (Shandon, Fisher Scientific) instructions; hematoxylin incubation of 12 min and eosin incubation of 4 min.
      For anti-CD68 immunohistochemical (IHC), deparaffinization, antigen retrieval and staining were performed on the automated Discovery Ultra staining system (Roche). For CD68 staining, antigen retrieval was done using the Discovery Ultra system with the CC1 protocol (cell conditioning buffer containing Tris–Borate-ethylenediamine tetraacetic acid, pH 8.0 and incubated for 88 min at 100°C). Following antigen retrieval, A/B block (Roche #760-050) and S block (Roche #760-4212) steps were performed. The primary antibody, rabbit polyclonal anti-CD68 (Abcam # ab125212), was used at a dilution of 1:50 in antibody dilution buffer (Roche ABD250) and applied for 60 min at 37°C. The secondary antibody was biotinylated goat antirabbit IgG (Rb Link Biogenex # HK3369r) and was applied for 32 min at 37°C. Staining was completed using a DABMap detection kit (Roche # 760-124), hematoxylin counterstain (Roche #760-2021), and bluing agent (Roche # 760-2037). “No-primary” antibody controls were run for each sample; for these slides, only antibody dilution buffer was applied at the primary antibody incubation step. Sections stained with H&E and anti-CD68 were scanned with an Aperio ScanScope XT (Aperio Technologies) and analyzed and photographed using Aperio Imagescope software.

      Statistical analyses

      All statistical analyses were performed using GraphPad Prism 9 (GraphPad Software). Data distribution normality was evaluated using the Shapiro–Wilk test for small groups (four animals per group) and D'Agostino-Pearson test for groups with seven or more animals. Subsequently, data were evaluated with nonparametric Kruskal–Wallis test in combination with Dunn's post-test for multiple comparisons or the Mann–Whitney U test when comparing only two groups. Uninfected mice were excluded from CFU statistical analysis because they primarily served as a control group for surgical procedures and baseline assessment of ambulation.

      Results

      Daptomycin treatment reduces infection when initiated prophylactically versus therapeutically

      In this study, we introduce a mouse model of surgical mesh infection. To approximate retro-rectus and inguinal surgical mesh placement in human hernia repair, we selected an easily accessible location in the upper leg of the mouse that had close contact with fascial planes of muscle and associated connective tissues (Fig. 1). Subsequently, the efficacy of therapeutic and prophylactic administration of antibiotic and/or anti-CD47 immunotherapy to treat S aureus infection was determined in this surgical mouse infection model following mesh implantation. Drug administration was initiated either immediately after surgery (prophylactic) or 3 days postimplantation (therapeutic) and treatments were continued for 10 consecutive days (Fig. 2). Administration of a bactericidal antibiotic (i.e., daptomycin) at the time of surgery is considered a prophylactic treatment in our animal model because it is given prior to bacterial attachment and infection of the mesh and prior to clinical signs. In our pilot studies, bacterial attachment on the surface of the mesh implant was observed at 3 days postinfection (dpi). Hence, we opted to initiate our therapeutic treatment on day 3 postsurgery. Groups of mice were euthanized at either 11 or 21 dpi for evaluation of bacterial loads on explanted mesh. Controls included mice receiving mock infection (uninfected) and S aureus–infected mice that were not treated (No Tx). Ambulation and use of the affected limb were scored on a scale of zero to three every 24 h in a blinded manner and median values are shown in Figure 2A and D. For all the experiments, mice that received mock infection had normal ambulation within a day following surgery, whereas untreated, infected mice developed variable levels of lameness in the left hind leg (Fig. 2A and D). Although the medians of the daptomycin and daptomycin plus anti-CD47 groups were similar, only the group that received antibiotic alone had ambulation scores significantly different (P ≤ 0.0228) from infected, untreated controls until day 12 of infection (Fig. 2A). Of note, this study was intended as a pilot experiment that allowed us to screen multiple treatment options and larger group sizes should be evaluated in future experiments. Ambulation score results were supported by enumeration of bacteria recovered from excised surgical mesh 11 days postsurgery. Groups that received daptomycin treatment reduced MRSA infections remarkably better than untreated controls, decreasing recovered CFUs from approximately 5 × 106 to 5 × 102 (Fig. 2B). The addition of anti-CD47 treatment did not improve control by daptomycin alone. By 21 dpi, all but one mouse treated with daptomycin alone or in combination with anti-CD47 cleared infections to an undetectable level (Fig. 2C).
      Figure thumbnail gr2
      Fig. 2Prophylactic treatment initiated at the time of surgery shows better clinical outcome over treatment started 3 days postsurgery. Groups of Y10 mice underwent mesh implantation surgery and 107 CFU S aureus instillation (n = 11) or saline instillation for uninfected controls (n = 5). Drug administration began either immediately after surgery (Prophylactic, panels A-C) or 3 days postimplantation (Therapeutic, panels D-F). All treatment regimens were continued for 10 days. Animals were scored daily using the following criteria: 0 = normal gate, 1 = slight limp, 2 = intermittent weight bearing, 3 = nonweight bearing, and the median values for each group at each time point are shown in A and D. Bacterial counts within excised infected mesh were analyzed on day 11 (B and E) or day 21 (C and F) postinfection and individual values with median and interquartile range are shown. CFU counts are shown as log-transformed values. Statistical analysis was performed using the Kruskal–Wallis test with Dunn's post-test for multiple comparisons. ∗ indicates P value < 0.05 for the daptomycin treatment group versus infected untreated control group. The median values for the combination treatment group (daptomycin + α-CD47) and the daptomycin alone were nudged for visibility.
      To determine the efficacy of therapy once bacterial infections had time to establish, antibiotic and/or anti-CD47 antibody treatment was tested beginning at 3 days postimplantation of contaminated mesh (Fig. 2D-F). All mice in all groups implanted with contaminated mesh showed impaired ambulation for 2 weeks following surgery (Fig. 2D). After this point, mice in all treatment groups appeared to have improved ambulation compared to untreated controls (Fig. 2D), but these changes were not statistically significant based on calculations performed with limited group sizes and multiple comparisons. In addition, there was no statistically significant reduction in bacterial loads in any of these groups on day 11 or day 21 relative to untreated controls (Fig. 2E and F). Thus, therapy beginning 3 days postinfection had little or no therapeutic effect.

      Single-dose daptomycin at the time of surgery

      Antibiotic prophylaxis with a single dose of antibiotics at the time of surgery is a common practice during surgical implantation of mesh, usually administered before surgical incision. To investigate the efficacy of prophylaxis in our model, a single dose of daptomycin was administered immediately after mesh implantation and S aureus infection. As previously, the mice were observed daily and ambulation scores were recorded. Results showed a clear improvement in use of the affected limb after a single daptomycin treatment compared to no treatment controls (Fig. 3A). At days 3, 11, and 21, mice from each group were euthanized for bacterial load analysis of excised mesh (Fig. 3B). After 3 dpi, MRSA CFU counts were reduced by approximately two logs compared to untreated controls (Fig. 3B) with an even greater decrease by day 11. By day 21, no bacteria were recovered from excised mesh in seven of eight animals treated with a single dose of antibiotic. Thus, a single dose delivered at the time of surgery appeared as effective as daily therapy for 10 consecutive days (compare Fig. 2, Fig. 3B, Fig. 2, Fig. 3C and, Fig. 2, Fig. 3B).
      Figure thumbnail gr3
      Fig. 3Single dose of daptomycin administered at the time of surgery offers sufficient protection from S aureus infection in mouse model of mesh implant. Groups of Y10 mice underwent mesh implantation surgery and 107 CFU S aureus instillation. Designated group of mice received a single dose of daptomycin (250 μg, Dapt) immediately after surgery or were left untreated (No Tx). (A) Median value of daily ambulation scores. Scoring criteria and methods are described in Material and Methods section. (B) Bacterial loads within excised mesh implants were evaluated on day 3, 11, and 21 postinfection. CFUs are shown as log-transformed individual values and median and interquartile range is indicated. Statistical analysis was performed using the Mann–Whitney U test. Ambulation score P value was calculated for each day separately and does not exceed P = 0.0014.

      H&E and immunohistological staining suggest an inhibition of macrophage infiltration into mesh site

      To visualize the structure and organization of the tissue pathology associated with placement of S aureus–infected mesh, we excised the entire upper leg of the mouse and tissue sections were stained with H&E. During tissue sectioning, the polypropylene mesh generally falls out of the section, leaving a spherical void in the tissue (examples are indicated with the letter ‘m’ in Fig. 4). H&E staining of a tissue sample from an uninfected mouse at 11 dpi (Fig. 4A) and a daptomycin-treated mouse at 21 dpi (Fig. 4B) showed that the surgical mesh became well incorporated into the surrounding connective tissues. A black dotted line in Figure 4 panels A and B shows the approximate borders of the mesh and resulting incorporation into the adjacent tissue. IHC staining for macrophages using an anti-CD68 antibody at high magnification showed CD68-positive cells around the perimeter of the mesh in both the uninfected (Fig. 4C) and the daptomycin-treated mouse (Fig. 4D).
      Figure thumbnail gr4
      Fig. 4H&E and anti-CD68 staining showing macrophage infiltration and response to uninfected and infected mesh. (A, B, and E) Representative H&E overview images from mesh implantation sites. The extent of the mesh implant and resulting fibrosis and incorporation into the surrounding tissues is demarcated with dashed black lines. Examples of where mesh was present are indicated with the letter “m”. (C, D, G, and I) Anti-CD68 immunohistochemistry demonstrating macrophage distribution (brown). In panels A, B, E, and G, the rectangles and associated letter correspond to panels that provide higher magnification of the outlined region. Tissue samples with mesh implantation site were collected from: (A and C) An uninfected mouse at 11 days postimplantation; (B and D) A daptomycin-treated mouse at 21 days postimplantation. (E-I) An untreated mouse with a large abscess present at 11 days post-MRSA infection. In panels F and G, the area shown includes the interior of the abscess adjacent to the mesh indicated with the letter “a”, an acellular region of abscess (b), and an organized abscess wall (c). Note, in panels C and D there is an abundance of macrophage (brown) near the mesh of uninfected and daptomycin-treated mice. In contrast, in panel G, the macrophages are present primarily at the perimeter of the abscess in untreated mice, far from the mesh. A no-primary antibody control to demonstrate the specificity of the CD68 staining is shown in panel (H) The scale bar in panel A applies to panels A, B, and E and represents 1 mm. The scale bar in panel C is 50 μm and applies to panels C, D, H, and I. The scale bar in panel F is 100 μm and applies to panels F and G.
      Mice that were untreated developed large abscesses surrounding the infected mesh (Fig. 4E). The dotted black line delineates the approximate border of the resulting abscess. Higher power magnification of the area in the black rectangle shows the abscess interior containing numerous neutrophils and bacteria (a), a region of amorphous, acellular debris and necrosis (b), and a highly cellular perimeter composed of fibrin, fibroblasts, and macrophages (c) (Fig. 4F). Sections from mice that received anti-CD47 treatment alone appeared similar to untreated mice (data not shown).
      IHC staining using anti-CD68 antibody was used to confirm the identity of macrophages, which were found around the perimeter of the abscess. Unlike the uninfected or daptomycin-treated mice, the mice with abscesses showed very few or no CD68-positive cells near the mesh (Fig. 4G). A higher magnification of the boxed area in panel 4G is shown in panel 4I to better demonstrate the macrophage morphology of CD68-positive cells (brown). Panel 4H is the no-primary antibody staining control from the same region as shown in 4I.

      Discussion

      Infection of surgical mesh is one of the most feared and difficult to treat complications associated with hernia repair surgery. In a recent study of patients who had to undergo reoperation for mesh explantation, median hospital costs approximately doubled, nearly half of the patients suffered further incisional complications, and the majority suffered hernia recurrence requiring an additional readmission.
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      Costs and complications associated with infected mesh for ventral hernia repair.
      Development of strategies to prevent and treat mesh infection is needed, but even simple questions such as "should antibiotic prophylaxis be used in mesh hernia surgery?" are difficult to answer in patient populations due to low rates of infection and the resulting difficulty in properly powering randomized control trials.
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      Some clinical studies to assess the benefit of antibiotic prophylaxis prior to mesh implantation have suggested benefit, but others have not, and there are no unequivocal data that have led to a consensus for standard practice (reviewed in
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      ). It is an important question because unnecessary antibiotic use is not only expensive but can lead to allergic reactions, antibiotic resistance, and disruption of the gut microbiome leading to Clostridioides difficile colitis. The results from our study demonstrating the high efficacy of antibiotic prophylaxis and the poor efficacy of treating established mesh infections are consistent with the trend in randomized control trials.
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      • et al.
      Meta-analysis of the effectiveness of prophylactic antibiotics in the prevention of postoperative complications after tension-free hernioplasty.
      This encourages us that the model is applicable, relevant, and useful in obtaining preclinical data for questions difficult to address in humans. Interfascial thigh placement of mesh was chosen to approximate both inguinal and retro-rectus mesh placement in humans. In these locations, both sides of the mesh are in apposition to a combination of muscle, fascia, and muscular aponeurosis. We felt that the proximity to muscle in our model would increase the chance of successful intervention. Intraperitoneal overlay mesh placement may differ in that the mesh is in apposition to a less vascular tissue, and as such, a more difficult area to clear infection. In this sense, we acknowledge that our model may not be entirely applicable to intraperitoneal mesh placement. However, all of the described locations have in common that mesh infection usually requires explantation for definitive control. It should be noted that in this model development study, only female mice were used. It has been demonstrated that gender-based differences could potentially influence the outcome of disease progression or therapy efficacy. Although in the presented animal model we cannot predict whether gender of the animals has an effect on disease progression or treatment, gender-based differences in immune response to infection exist.
      • McClelland E.E.
      • Smith J.M.
      Gender specific differences in the immune response to infection.
      Furthermore, there is evidence that male mice are more susceptible to S aureus skin and soft tissue infections.
      • Castleman M.J.
      • Pokhrel S.
      • Triplett K.D.
      • et al.
      Innate sex bias of Staphylococcus aureus skin infection is driven by α-hemolysin.
      ,
      • Pokhrel S.
      • Triplett K.D.
      • Daly S.M.
      • et al.
      Complement receptor 3 contributes to the sexual dimorphism in neutrophil killing of Staphylococcus aureus.
      We were interested in CD47 blockade because it has been shown that bacterial infections stimulate upregulation of host cell surface CD47, which has an inhibitory effect on macrophages and other immune cells.
      • Tal M.C.
      • Dulgeroff L.B.T.
      • Myers L.
      • et al.
      Upregulation of CD47 is a host checkpoint response to pathogen recognition.
      The blockade of CD47 results in enhancement of innate and adaptive responses against a broad range of infectious agents, including viruses and certain bacteria such as Mycobacterium tuberculosis.
      • Tal M.C.
      • Dulgeroff L.B.T.
      • Myers L.
      • et al.
      Upregulation of CD47 is a host checkpoint response to pathogen recognition.
      Thus, we investigated the potential for enhancing S aureus clearance by CD47 blockade alone or in combination with daptomycin. Interestingly, CD47 blockade on its own decreased clinical signs when administered as a therapeutic (Fig. 2D) but did not significantly improve bacteria clearance (Fig. 2E, F). It is possible that analysis of more animals might show statistically significant reductions at day 21 but those reductions would be slight and not change the conclusion that antibiotic therapy beginning at 3 days postsurgery was ineffective. Histological studies at 21 days showed that very few if any macrophages were present near the mesh when abscesses were present. This may have been due to the fact that S aureus produces an arsenal of immune evasion molecules and forms protective biofilms on the implant surfaces that promote immune escape, antibiotic resistance, and persistent infection.
      • Arciola C.R.
      • Campoccia D.
      • Montanaro L.
      Implant infections: adhesion, biofilm formation and immune evasion.
      We observed no significant benefit on either clinical signs or bacterial clearance when anti-CD47 was added to daptomycin prophylaxis or therapy. Although this particular immunotherapy failed, we think that we have developed a workable, preclinical platform to test new prophylactics, therapeutics, and different types of biologic and prosthetic mesh materials.

      Author Contributions

      All authors made substantial contribution to the study, critically reviewed, and gave final approval of the manuscript prior to submission. Conception and design of experiment was done by A.M.H., K.H., B.R., R.M., and N.M. M.C., B.R., R.M., C.B., D.L., K.W., and N.M. performed experiments and collected the data. M.C., B.R., R.M., S.D.K., K.H., and N.M. analyzed and interpreted the data. M.C., B.R., K.H., A.M.H., and N.M. wrote the manuscript.

      Acknowledgments

      We acknowledge important discussions with James F. Striebel (Laboratory of Persistent Viral Diseases, RML/NIH) on interpretation of results and graphics assistance from Anita Mora and Rose Perry-Gottschalk (Visual & Medical Arts, RML/NIH).

      Disclosure

      None declared.

      Funding

      This research was funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

      Data

      Raw data are available upon request.

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