Abstract
Indwelling foreign‐body infections are a critical medical problem, especially in immunocompromised patients. To examine the pathogenicity of biofilm‐forming bacteria settling on foreign materials, mice implanted with plastic discs were infected with Staphylococcus aureus. After opening a wide subcutaneous pocket on the dorsal side of mice with or without temporal leukocytopenia, a plastic sheet was placed in the left subcutaneous space; subsequently, bacteria in a planktonic state were dispersed over the subcutaneous space. Bacterial numbers were examined 7 days after inoculation. In subcutaneous tissue on the right, S. aureus was found only in leukocytopenic mice. Meanwhile, bacteria were detected on the plastic and neighbouring tissue in both leukocytopenic and normal mice; however, colony‐forming analysis indicated that leukocytopenic mice possessed significantly more bacteria. Tissue reaction against bacteria was pathologically examined. Invading S. aureus induced severe inflammation. In transient leukocytopenic mice, bacterial microcolonies formed on the plastic as well as in the developed necrotic tissue – both of which were shielded from inflammatory cell infiltration – result in bacteraemia. These results indicate that biofilm‐forming S. aureus settling on indwelling foreign material are tolerant against host immunity and assault neighbouring tissue, which may lead to chronic wound infection.
Keywords: Biofilm, Foreign materials, Immunosuppression, Mice, Staphylococcus aureus
Introduction
Approximately 45% of hospital‐acquired infections are caused by contaminated foreign materials or medical devices 1, 2. Staphylococcus aureus is one of the most frequently detected microbes in clinical practice and is found in abscesses, food poisoning, pneumonia and sepsis 3. S. aureus can easily acquire multiple drug resistance via spontaneous mutations and DNA transfer 4.
The effects of biofilms on wounds have been investigated in many clinical and animal studies 5, 6, 7, 8. Such studies conclude that biofilm is an aggravating factor in delayed wound healing. S. aureus is capable of forming biofilms by attaching to substrate surfaces. These biofilms are highly complex structures consisting of microbial cells embedded in extracellular matrices of hydrated extrapolymeric substances 9. In vivo, their tolerance properties against phagocytic cells and/or antibiotics are also believed to be exerted by their biofilms 10. Bacterial biofilms readily form on the non‐ biological substrates of medical devices including cannulas and prosthetic joints. Once formed, they are quite difficult to remove. Meanwhile, approximately 60% of chronic wounds involve microbe infection 11. Our previous study on chronic infectious wounds in patients shows that the thickness of the covered necrotic layer on granulation tissue increased in accordance with increased infecting S. aureus number 12. In chronic wounds, scaffold matrixes and/or special microenvironments that allow bacteria to form biofilms may emerge in wounded tissue.
It is well known that infectious risk increases in patients with diabetic mellitus and immune deficiency as well as in those on chemotherapy. Several animal studies clearly show that S. aureus forms more number of microcolonies in wounded tissue in neutropenic animals pre‐treated with an immunosuppressive drug 5, 6, 8. Specific conditions required for bacteria to be immune to the attack of inflammatory cells could be necessary for biofilm formation in living tissue. However, how local infection can initiate the development of chronic wounds remains unclear.
In this animal study, we induced a marrow‐suppressive and leukopenic state in mice by 5‐fluorouracil (5‐FU) pre‐treatment, subcutaneously indwelled them with a piece of plastic as a foreign material and inoculated them with biofilm‐forming S. aureus. We investigated the in vivo pathogenicity of S. aureus biofilm on the plastic and clarified the roles of the bacterial biofilm in the development of chronic wounds.
Materials and methods
Animal care
C57BL6 female mice aged 6–10 weeks (Japan SLC Inc., Shizuoka, Japan) were used. The Animal Care Committee of Fukuoka University approved the study protocol.
Staphylococcus aureus
In routine bacterial examinations in the Fukuoka University Hospital, Fukuoka, Japan, methicillin‐resistant S. aureus (MRSA) colonies were isolated from patients with sepsis and/or wound infections. One colony was grown on an agar plate, and established as a strain. Of 175 MRSA strains, we selected one strain, OJ1, isolated from a 67‐year‐old patient treated for a leg ulcer due to an open fracture. OJ1 positive for S. aureus‐specific antibody (ViroStat Inc., Westbrook, ME) exhibits one of the greatest biofilm forming abilities, which was assessed by crystal violet staining of 10‐ml plastic tubes with Difco Tryptic Soy Broth (TSB; BD Co., Franklin Lakes, NJ) after 24 hours incubation at 37°C 13. However, the OJ1 colonies exhibited a normal appearance and did not differ from other strains. OJ1 stored at −80°C was cultured on Difco Tryptic Soy Agar (BD Co.), and one colony was obtained and cultured in TSB at 37°C. The absorbance of 10‐fold serially diluted S. aureus solution was measured with a spectrometer (Novaspec II, Pharmacia Biotech, Tokyo, Japan). The diluted S. aureus solutions were seeded on agar plates, and colony‐forming units (CFUs) were determined. Small numbers of S. aureus in TSB were cultured at 37°C, and increasing cell density was monitored with a spectrometer at 578 nm up to an optical density of 0·57 (i.e. 2 × 108 CFU/ml equivalent). Bacterial solution in a planktonic state on ice was used as a stock solution for the mouse infection study.
Model 1 – foreign‐body infection model
A circular plastic disc (OHP film, Kokuyo, Osaka, Japan) with a 1‐cm radius was used as a foreign material in vivo (Figure 1). Mice were pre‐treated with or without an intraperitoneal injection of 5‐FU (Sigma‐Aldrich Japan Co., Tokyo, Japan) for marrow suppression at a dose of 2·25 mg/100 µl·20 g per body weight (n = 5 in each group). The effect of 5‐FU on marrow suppression was determined by morphometric analysis (VH Analyzer, KEYENCE, Osaka, Japan). Under anaesthesia, a wide subcutaneous pocket was created by blowing air. A small incision was made on the dorsal skin, a plastic disc was inserted into the left side of the pocket and the wound was sutured. Three days after disc insertion, an OJ1 solution with 4 × 107 CFU (200 µl 2 × 108 CFU/ml) was inoculated into the whole pocket area by a syringe. After 7 days, the animals were euthanised, the pocket was opened, and the disc, tissues under the plastic disc on the left side and subcutaneous tissue on the right side were obtained. These samples were used for the CFU assay.
Figure 1.
Mouse infection models. Model 1 (foreign‐body infection model): this model was used to study the role of indwelling circular plastic sheets on the transmission of planktonic Staphylococcus aureus in mice with or without leukocytopenia. In brief, a wide subcutaneous pocket on the dorsum was created, and a 10‐mm circular plastic sheet was inserted into the left side on day 0. On day 3, S. aureus solution was dispersed over the whole pocket area. On day 7, a colony‐forming assay was performed on the peripheral blood, plastic, neighbouring tissue on the left and subcutaneous tissue on the right. Model 2 (infection wound model): this model was used to study the interaction between S. aureus and wound tissue in mice with or without leukocytopenia. Subcutaneous tissue exposed on the back was injured by 70% alcohol exposure, and the wounded tissue was covered by a 20‐mm circular plastic sheet set under the dorsal skin. After 3 days, planktonic S. aureus solution was inoculated between the plastic and the wound. Tissue samples were obtained every 2 days until day 10 of the study.
CFU assay of blood, plastic and wound tissue samples
Bacterial cultures for CFU assays were prepared. Blood (75 µl) from the orbit was collected aseptically by a haematocrit tube, and 40 µl blood was serially diluted 10‐ and 100‐folds with TSB. The plastic was aseptically removed from the body, and the accretive deposit on the plastic was swabbed with a cotton bud and soaked in 3·14 ml ice‐cold TSB. The solution was mixed vigorously and serially diluted 10‐fold up to 105 times. Granulation wound tissue beneath the plastic sheet was also collected aseptically (20 µg) in 200 µl ice‐cold 0·05 M phosphate‐buffered saline. The tissue was then homogenised by a micro‐homogeniser (Polytron PT 1200E, KINEMATICA AG, Lucerne, Switzerland); 50 µl solution was diluted 100‐fold with TBS, and serial 10‐fold dilutions were performed up to 107 times. Diluted tissue solution (50 µl) was seeded on agar plates, which were incubated overnight at 37°C. Colonies on agar plates were counted and expressed as log CFU per 10 mg tissue weight, per 10 ml blood and per square centimetre plastic.
Model 2 – infection wound model
For pathological investigation, we created another foreign‐body infection model with some modifications that may lead to chronic wounds (Figure 1). In brief, after disinfection of shaved back skin, a small circle 15 mm in diameter of full‐thickness skin was excised. The wound surface was then exposed to 70% ethanol for 1 minute for disinfection and wounding by dehydration. A circular plastic disc, 20 mm in diameter, was sterilised with 70% alcohol and placed on the dorsal muscle via insertion under the back skin and fixed with adhesive glue. In mice pre‐treated with or without 5‐FU, 4 × 107 CFU S. aureus solution (200 µl 2 × 108 CFU/ml) was injected using a syringe into the space between the plastic disc and wound surface on day 3.
All the mice used in this study underwent dorsal skin excision and plastic disc insertion on day 0 of the study. Mice were then divided into four groups (n = 25 per group): the untreated (control), 5‐FU pre‐treated (5‐FU), S. aureus‐seeded (SA) and 5‐FU pre‐treated and S. aureus‐seeded (5‐FU + SA) groups. The numbers of platelets, white blood cells (WBC) and red blood cells were routinely checked by an automatic haematocytometer (MEK‐6358, NIHONKOHDEN Co., Tokyo, Japan) during the study. Body weight on day 0 of the study was 16·4 ± 0·6 g, ranging from 15·0 to 17·5 g. Tissue under the plastic was obtained for histological analysis.
Histological examination
Mice were euthanised every 2 days (five time points), and five mice per group were examined histologically. Wound tissue was fixed in 10% buffered formaldehyde (pH 7·4), embedded in paraffin and cut into 3‐µm‐thick sections with a microtome. When much sediment accumulated on the plastic sheet, a procedure similar to that for tissue was performed. Then, serial sections were stained with haematoxylin−eosin, Gram's stain, periodic acid‐Schiff (PAS), alcian blue and acridine orange. The sections were also used for immunohistological examination. The sections were immersed in 1% bovine serum albumin (Sigma‐Aldrich Japan Co.) for 30 minutes and incubated for 1 hour at room temperature in a 1:100 dilution of rabbit S. aureus antibody (ViroStat Inc.), 1:500 dilution of rat Ly‐6 G/‐6C neutrophil antibody (Hycult Biotech, Uden, The Netherlands) and 1:200 dilution of rabbit macrophages (Mac‐1) antibody (LifeSpan BioScience Inc., Seattle, WA). The EnVision Kit (DAKO Japan Inc., Kyoto, Japan) and rat ABC staining system (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used for visualisation. The plastic sheets removed from the mice were also used for histological examination. The plastic sheets were fixed in 10% buffered formaldehyde. After washing in phosphate‐buffered saline, the plastic sheets were stained in the same manner as the tissue sections.
Transmission electron microscopy
The plastic sheet was fixed with 2% glutaraldehyde, post‐fixed with osmium tetroxide and embedded in Epon resin. Ultrathin sections were double stained with lead citrate and uranyl acetate and observed under a transmission electron microscope (JEM‐1200 EX, JEOL, Tokyo, Japan).
Statistical analysis
All data are expressed as mean ± SEM. Differences between groups were examined for statistical significance using Student's t‐test and one‐way analysis of variance (ANOVA). A P value <0·05 was considered statistically significant.
Results
Roles of indwelling plastic sheets in infection
In this study, we used 5‐FU, an anti‐tumour drug known to induce bone marrow suppression. The dose used in this study induced a temporal decrease in marrow cell number on days 2–4 (Figure 2A); however, cell numbers recovered from day 6.
Figure 2.
Colony‐forming analysis in model 1 study. (A) Number of bone marrow cells in control mice (empty column) and mice with 5‐fluorouracil (5‐FU) pre‐treatment (closed column) were histologically counted every 2 days. (B) Colony‐forming analysis for mice inoculated with Staphylococcus aureus (empty column) and 5‐FU pre‐treated mice inoculated with S. aureus (closed column). Samples on day 10 were obtained from the plastic sheet, neighbouring tissue on the left and subcutaneous tissue on the right. Blood samples for colony‐forming unit (CFU) were obtained every 2 days. Data are expressed as mean ± SEM.
In model 1, a CFU assay was performed on day 10 in mice pre‐treated with or without 5‐FU. In mice pre‐treated without 5‐FU (Figure 2B, SA group), no S. aureus was found in tissue without plastic (right tissue). However, the plastic sheet and its neighbouring tissue (left tissue) had bacteria. In mice pre‐treated with 5‐FU (Figure 2B, 5‐FU + SA group), all samples from the three different areas had significantly larger numbers of S. aureus than the SA group. Peripheral blood was also used for the CFU assay. Almost no bacteria were found in the SA group during the study. While in the 5‐FU + SA group, bacteria were detected on day 6; however, they appeared on day 8 and decreased on day 10, showing transient bacteraemia.
Physiological responses against foreign‐body infection
In the presence of the plastic sheet, the relationship between marrow suppression and S. aureus infection was investigated using model 2 mice. About 10–15% body weight loss was observed after bacterial inoculation in the 5‐FU + SA group during the study (Figure 3A). However, a single treatment of 5‐FU or bacterial inoculation induced only a slight decrement on day 4 alone.
Figure 3.
Changes in the body weight and white blood cells (WBC) over time. In model 2, the mice were divided into four groups: the control, 5‐fluorouracil pre‐treated (5‐FU), Staphylococcus aureus‐seeded (SA) and 5‐FU pre‐treated and S. aureus‐seeded (5‐FU + SA) groups. Body weight (A) and WBC count (B) were monitored every 2 days. Data are expressed as mean ± SEM.
The WBC count was monitored to investigate haematological alterations during inflammation against wounding and infection (Figure 3B). Among the four groups, WBC count increased significantly in the control group on day 6, and the largest decrease was noted in the 5‐FU + SA group on day 4. Compared with the control group, a single treatment of 5‐FU or bacterial inoculation lowered the WBC levels.
Bacterial settlement on the plastic sheet
After unfixing the plastic sheet from the back, bacteria on the plastic sheet were immunohistochemically stained with a S. aureus‐specific antibody. The chromomeric area positive for S. aureus was measured (Figure 4A). On day 4, no difference was observed between the SA and 5‐FU + SA groups. However, the area gradually increased in the 5‐FU + SA group, whereas it decreased in the SA group; the difference between the groups was significant on day 10.
Figure 4.
S. aureus settled on plastic sheets. Plastic sheets in model 2 were obtained and used for bacterial density analysis and electron microscopy. (A) Bacterial density analysis was performed morphometrically with immunohistological staining using an antibody against S. aureus. Data are expressed as mean ± SEM. (B) Ultrastructural examination of plastic sheets from mice in the 5‐FU pre‐treated and S. aureus‐seeded (5‐FU + SA) group on day 10. There are two distinctive layers on the plastic sheet: a colony layer that contains many bacterial microcolonies (*) surrounded by membranous/amorphous structure (arrows) and a necrotic cell mass that covers the colony layer. Most bacteria are found in dying phagocytic cells. Note: Inflammatory cells including phagocytes were found in this layer, but not in the colony layer.
Biofilm formation on the plastic sheet
S. aureus that settled on the plastic sheet on day 10 in the 5‐FU + SA group were examined by electron microscopy (Figure 4B). S. aureus microcolonies formed directly on the plastic surface and were demarcated by a thin membranous structure and an amorphous electron‐lucent matrix (Figure 4B, bottom right). However, no inflammatory cells were noted inside of the biofilm. Meanwhile, a thick necrotic cell mass with numerous phagocytotic cells ingesting round bacteria accumulated beneath the biofilm (Figure 4B, bottom left).
Staphylococcus aureus and wound tissue reaction
On day 10, the appearance of wounds through the plastic sheet exhibited distinct features in each group (Figure 5A): mice in the control and 5‐FU groups showed a red colour, mice in the SA group showed a xanthous colour and mice in the 5‐FU + SA group showed a characteristic white‐ringed deposition with purulent matter.
Figure 5.
Alterations in the wound tissue due to S. aureus infection. (A) Representative features of the wound through the plastic window in the four groups. (B) Representative tissue images of wound surface under the plastic sheet. Natural course of wound healing in the control group; inflammation was noted from day 4, and granulation (*) started from day 6. In the 5‐fluorouracil pre‐treated (5‐FU) group, no inflammation was found until day 6, but granulation (*) started from day 8. In the S. aureus‐seeded (SA) group, severe inflammation started from day 4 but gradually decreased thereafter. In the 5‐FU pre‐treated and S. aureus‐seeded (5‐FU + SA) group, microcolonies (arrows) formed and severe inflammation under the colonies started from day 8.
Figure 5B shows representative images of histological changes in the wounds. Wound healing took a natural course in the control group. Inflammatory cell infiltration began on day 4 and increased on day 6; it subsequently regressed, and a thin granulation tissue developed on the surface on day 10. Such inflammatory cell infiltration was low on days 4–6 in the 5‐FU group; however, granulation tissue developed on day 10 to a similar extent as that in the control group. In the SA group, severe wound inflammation occurred 1 day after S. aureus inoculation (day 4); this was prolonged until day 8 but subsequently regressed accompanied by granulation tissue development on day 10. In the 5‐FU + SA group, many invading and proliferating S. aureus aggregates formed microcolonies near the wound surface until day 6. Severe inflammatory cell accumulation, which formed a necrotic cell mass accompanied by microcolony distortion, occurred on day 8 and intensified on day 10.
Staphylococcus aureus invasion and inflammatory cells
In the 5‐FU + SA mice, microcolony‐forming S. aureus gradually invaded the muscle layer from the wound surface (Figure 6A). On day 10, S. aureus discharged from the microcolonies were occasionally observed (Figure 6B), manifesting as a partially disconnected Schiff‐positive polysaccharide‐containing membranous structure around the colony.
Figure 6.
Microcolony invasion in the wound tissue. (A) The extent of S. aureus microcolonies in wound tissue was compared using Gram‐stained samples on days 4 and 10 in the 5‐FU pre‐treated and S. aureus‐seeded (5‐FU + SA) group. Microcolonies localised at the surface on day 4 invaded deep into the wound on day 10. (B) To examine microcolony invasion in detail, tissues were double stained by Gram's stain and periodic acid‐Schiff (PAS). In a sample from the 5‐FU + SA group on day 10, discharged S. aureus from one of the microcolonies (*) is seen, where a Schiff‐positive polysaccharide‐containing membranous structure around the colony was partially disconnected (arrows).
Next, the relationship between S. aureus invasion and inflammatory cells was investigated using wound samples from the SA and 5‐FU + SA groups on days 4 and 10. The sample on day 4 in the SA group shown in Figure 7 indicates that scattered bacteria were present in the shallow area of the wound surface and colocalised with neutrophils and macrophages, which are responsible for most phagocytosis. However, on day 10, the bacteria mostly disappeared and inflammatory cells decreased.
Figure 7.
Distribution of S. aureus in the wound tissue. Time course of the distinct distributions of S. aureus, neutrophils and macrophages (Mac‐1) in the S. aureus‐seeded (SA) and 5‐FU pre‐treated and S. aureus‐seeded (5‐FU + SA) groups are shown. Immunostainings (brown) are counter‐stained with haematoxylin (blue). In the SA group on day 4, the dispersed S. aureus (arrows) were accompanied by infiltrating inflammatory cells including neutrophils and macrophages; most bacteria are phagocytised, and S. aureus and inflammatory cells decreased in number on day 10. In the 5‐FU + SA group on day 4, microcolonies (*) formed on the line of the wound surface but inflammatory cells were scarce; on day 10, microcolonies (*) with different sizes grew in the necrotic mass area framed by the red line, but no infiltrating inflammatory cells were observed. In contrast, severe inflammation (arrows) was noted beneath the colonies, where phagocytised S. aureus was colocalised with neutrophils and macrophages.
In the 5‐FU + SA sample on day 4 (Figure 7), bacteria formed microcolonies on the line of the wound surface but no inflammatory cells appeared because of the leukocytopenia induced by 5‐FU. On day 10, a distinctive necrotic mass (framed by the red line) was localised in the upper part of the wounded tissue, in which many proliferating microcolonies were observed; however, no inflammatory cell infiltration was noted. In contrast, in the portions beneath the red zone, microcolonies invaded beyond the muscle layer. Huge numbers of macrophages and neutrophils accumulated underneath the colonies, which ingested a number of bacteria.
Discussion
It is well known that bacterial biofilms formed on indwelling medical devices induce critical problems in patients. In this animal study, we clearly showed that transit immunosuppression and indwelling foreign‐body infections are two important pathogenic factors for the development of chronic wounds. Furthermore, biofilms that form on foreign bodies are tolerant against host immunity.
Biofilms formed by microbes including S. aureus comprise polysaccharides, proteins, nucleic acids and fibrin 14. Small amounts of PAS‐positive substances were sometimes found on the wound surface and plastic sheets in uninfected normal mice. Therefore, the PAS‐positive substances are not limited to biofilm; further, they include endogenous polysaccharides present in the wound, such as glycosaminoglycans 15, glycoproteins and glycogen. On the other hand, S. aureus colonies were usually surrounded by a thin polysaccharide‐positive membrane‐like structure. We applied different staining methods including PAS, alcian blue, acridine orange, concovalin A‐FITC and commercially available biofilm‐staining fluoresce dyes (data not shown). Only PAS and acridine orange staining highlighted the membrane‐like structure around the S. aureus colonies. Electron microscopy also showed that the demarcated wall of the S. aureus colonies consisted of basement membrane‐like materials. In this study, many invading inflammatory cells were visible at the disrupted sites of the outer membrane of the colonies where invading bacteria were phagocytised. These findings show that the demarcating structure of bacterial colonies works as the enveloping membrane of S. aureus colonies by which bacteria may be structurally protected against inflammatory cell attacks.
The effects of bacterial biofilms on wounds have been investigated in many animal studies. Schierle et al. 16 established an infected splinted mouse model in which they inoculated open wounds with biofilm‐ and non‐ biofilm‐forming bacteria; they found that the biofilm delayed wound reepithelialisation. A significant delay in wound healing was reported in an infected wound model of diabetic db/db mice with Pseudomonas aeruginosa biofilm on a filter 17. Furthermore, a wound evaluation model using rabbit ears showed that S. aureus rapidly forms biofilm in wounds 24 hours after inoculation 18. All of these studies conclude that biofilm is an aggravating factor in delayed wound healing. However, the in vivo mechanisms of the biofilm development remain unclear.
Akiyama et al. 5, 6 report that biofilm‐forming S. aureus inoculated on cutaneous wounds form more number of microcolonies in mice treated with cyclophosphamide as an immunosuppressive drug. In this study, we used a low dose of 5‐FU for a similar purpose. It is known that 5‐FU inhibits nucleic acid synthesis and its half‐life is quite short (10–20 minutes) 19. Therefore, a single shot of 5‐FU could induce a transient anti‐proliferative effect on marrow cells. The brief leukocytopenic state induced during S. aureus inoculation was sufficient for biofilm formation on the plastic sheets. The area of aggregated S. aureus on the plastic sheet gradually increased with time (Figure 4A). In contrast, the area gradually decreased in the SA group. The data recorded on day 4 in Figure 4A appear to show the reverse. However, it is possible that, in the SA group, many phagocytes on the plastic may have gathered and scavenged the inoculated bacteria. The tissue inflammatory response against S. aureus infection was completely different between the control and 5‐FU pre‐treated mice. Therefore, even a short‐term neutropenic state in vivo could be crucial for biofilm formation on indwelling foreign bodies. In the 5‐FU + SA group, the WBC level decreased on days 4–6 and was accompanied by significant body weight loss; moreover, bacteraemia appeared on day 8. However, such critical conditions did not manifest in the SA group. This difference may be attributable to in vivo biofilm formation.
Although S. aureus colonies were scarce in the SA group, dispersed S. aureus were widely distributed near the wound surface; these bacteria were mostly phagocytised by densely accumulated inflammatory cells. S. aureus can secrete many types of exotoxins including enterotoxins A–E, toxic shock syndrome toxin‐1, exfoliative toxins and leukocidin 20, which may have led to the formation of the necrotic cell mass observed in the 5‐FU + SA group.
In this study, the accumulation of inflammatory cells within the tissue indicated the presence of S. aureus in both the SA and 5‐FU + SA groups. However, they were not identical. In the SA group, phagocytes with bacteria were found in developing granulation tissue. However, in the 5‐FU + SA group, phagocytes with bacteria were present in dying and/or necrotic tissue with bacterial colonies and strongly expressed apoptotic cell markers (data not shown).
On the other hand, the colonies formed by aggregated S. aureus were found on the plastic sheets (Figure 4B) as well as in the necrotic mass on day 10 in the 5‐FU + SA group (Figure 7), in which inflammatory cells were rare. These results indicate that in vivo, the necrotic mass may act as a foreign body for S. aureus. Therefore, it is possible that bacteria settled on an indwelling foreign body subsequently invade the neighbouring necrotic tissue and form a biofilm again. Furthermore, this process may accelerate the development of chronic wounds.
In conclusion, we examined the relationship between bacterial distribution and its induced inflammation response in vivo after inoculating biofilm‐forming S. aureus into mice implanted with a foreign material. The possible process is shown in Figure 8: biofilm‐forming S. aureus settling on indwelling foreign material are tolerant against host inflammatory cells, assault neighbouring tissue and form necrotic tissue. Even if the foreign material is removed at this time, bacteria can form a biofilm in the necrotic tissue, which acts as a sort of new foreign material. Such infected wounds may become chronic wounds that are difficult for antibacterial agents and host immunity to resolve. Therefore, the destruction of biofilms formed in tissue is necessary to eradicate S. aureus infection.
Figure 8.
S. aureus infection and chronic wound. A schematic illustration of in vivo reactions against S. aureus is shown. If immunocompromised patients with indwelling foreign material are infected with S. aureus, the bacteria easily form a biofilm on the foreign material and invade neighbouring tissue, forming necrotic tissue. Even if the foreign material is removed, bacteria can still form a biofilm on the necrotic tissue, which acts as a new foreign material. Such infected wounds may become chronic wounds that are difficult for antibacterial agents and host immunity to resolve.
Acknowledgements
The authors express their sincere gratitude to Mr. Hisashi Kurata and Ms. Hitomi Manabe for their excellent technical help. All authors declare no conflict of interest in this study.
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