脊柱结核内固定的生物学基础

目的观察结核杆菌与不同内固定材料的粘附情况,以期初步探讨在脊柱结核病灶清除后植入内固定选择何种材料的问题。方法在结核杆菌的菌液分别加入四种不同的内固定材料（不锈钢、钛、钛合金、聚醚醚酮）,通过碘-125标记测量,比较细菌与材料之间的粘附情况。结果四种材料中,细菌在聚醚醚酮上的粘附最多,在纯钛上粘附最少,在不锈钢的粘附多于钛合金（P〈0.05）。结论在脊柱结核患者内固定材料的选择上,以纯钛为首选,钛合金次之,碳纤维材料慎用。     

Mechanical failure of hydroxyapatite- and polysulfone-coated titanium rods in a weight-bearing canine model.

Three types of material that have shown potential as coatings for orthopaedic implants were studied. Using a weight-bearing canine model, Ti-6A1-4V femoral intramedullary rods coated with (1) sintered titanium beads, (2) plasma-sprayed hydroxyapatite, and (3) silyl coupled polysulfone beads were evaluated for mechanical strength and bone ingrowth. The model was designed to secure optimal prosthetic stability by obtaining maximal bony ingrowth during an initial non-weight-bearing phase, then stressing the implant during a full-weight-bearing phase. None of the rods coated with titanium beads failed. All 17 polysulfone-coated rods failed, 13 of them at the interface between the polysulfone coating and the titanium core. Of 18 rods coated with hydroxyapatite, 15 suffered implant breakdown at the interface between the hydroxyapatite coating and the titanium core. This may be due to dissolution of the plasma-sprayed hydroxyapatite in vivo. Testing of retrieved specimens from both hydroxyapatite- and polysulfone-coated implants showed that the shear strength at the coating-rod interface had decreased to less than 40% of the shear strength at manufacture. Despite mechanical failure, histologic study showed extensive bone ingrowth or apposition onto both the polysulfone and hydroxyapatite coatings.     

Adherent lipopolysaccharide inhibits the osseointegration of orthopedic implants by impairing osteoblast differentiation

Osseointegration is the process by which an orthopedic implant makes direct bone-to-implant contact and is crucial for the long-term function of the implant. Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopedic implants after sterilization and impair osseointegration. For example, specific lots of implants that were associated with impaired osseointegration and high failure rates were discovered to have contaminants including bacterial debris. Therefore, the goals of this study were to determine if bacterial debris exists on sterile orthopedic implants and if adherent bacterial debris inhibits the osseointegration of orthopedic implants. We found that debris containing lipopolysaccharide (LPS) from Gram-negative bacteria exists on both sterile craniofacial implants and wrist implants. Levels of bacterial debris vary not only between different lots of implants but within an individual lot. Using our murine model of osseointegration, we found that ultrapure LPS adherent to the implants inhibited bone-to-implant contact and biomechanical pullout measures. Analysis of osseointegration in knock-out mice demonstrated that adherent LPS inhibited osseointegration by signaling through its primary receptor, Toll-like receptor 4, and not by signaling through Toll-like receptor 2. Ultrapure LPS adherent to titanium alloy discs had no detectable effect on early stages of MC3T3-E1 osteogenesis in vitro such as attachment, spreading or growth. However, later stages of osteogenic differentiation and mineralization were inhibited by adherent LPS. Thus, LPS may inhibit osseointegration in part through cell autonomous effects on osteoblasts. These results highlight bacterial debris as a type of surface contaminant that can impair the osseointegration of orthopedic implants.     

Effects of photodynamic laser and violet-blue led irradiation on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilm and <Emphasis Type="Italic">Escherichia coli</Emphasis> lipopolysaccharide attached to moderately rough titanium surface: in vitro study

Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This study investigates the in vitro efficacy of photodynamic treatment (PDT) with methylene blue (MB) photoactivated with λ 635 nm diode laser and of λ 405 nm violet-blue LED phototreatment for the reduction of bacterial biofilm and lipopolysaccharide (LPS) adherent to titanium surface mimicking the bone-implant interface. Staphylococcus aureus biofilm grown on titanium discs with a moderately rough surface was subjected to either PDT (0.1% MB and λ 635 nm diode laser) or λ 405 nm LED phototreatment for 1 and 5 min. Bactericidal effect was evaluated by vital staining and residual colony-forming unit count. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, discs coated with Escherichia coli LPS were treated as above before seeding with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Both PDT and LED phototreatment induced a statistically significant (p?<?0.05 or higher) reduction of viable bacteria, up to ?99 and ?98% (5 min), respectively. Moreover, besides bactericidal effect, PDT and LED phototreatment also inhibited LPS bioactivity, assayed as nitrite formation, up to ?42%, thereby blunting host inflammatory response. Non-invasive phototherapy emerges as an attractive alternative in the treatment of peri-implantitis to reduce bacteria and LPS adherent to titanium implant surface without causing damage of surface microstructure. Its efficacy in the clinical setting remains to be investigated.     

Cell-mediated bioresorption of sintered carbonate apatite in rabbits

Bone apatite contains carbonate and is therefore not pure hydroxyapatite. We have successfully developed sintered carbonate apatite (CA) with a concentration of carbonate of 6 weight% and have evaluated its osteoconductive and bioresorption characteristics. Cylindrical porous sintered CA and sintered hydroxyapatite (HA) measuring 4 x 4 mm with a porosity of 20% were implanted into surgically-created bone defects in the knees of rabbits. The animals were killed after 1, 3, 6 and 12 months. The defects were evaluated by microfocus CT and histology. Bone growth into and around both materials increased. Newly-formed bone was placed in direct contact with both. Osteoclast-like cells resorbed only CA, and were coupled with osteoblasts. The porosity of sintered CA increased, indicating bioresorption, whereas that of sintered HA did not increase. Our findings indicate that sintered CA may be useful as a bioresorbable bone substitute.     

Bacterial adherence to tantalum versus commonly used orthopedic metallic implant materials

OBJECTIVES: Evaluation of bacterial adhesion to pure tantalum and tantalum-coated stainless steel versus commercially pure titanium, titanium alloy (Ti-6Al-4V), and grit-blasted and polished stainless steel. DESIGN: Experimental in vitro cell culture study using Staphylococcus aureus and Staphylococcus epidermidis to evaluate qualitatively and quantitatively bacterial adherence to metallic implants. METHODS: A bacterial adhesion assay was performed by culturing S. aureus (ATCC 6538) and S. epidermidis (clinical isolate) for one hour with tantalum, tantalum-coated stainless steel, titanium, titanium alloy, grit-blasted and polished stainless steel metallic implant discs. Adhered living and dead bacteria were stained using a 2-color fluorescence assay. Adherence was then quantitatively evaluated by fluorescence microscopy and digital image processing. Qualitative adherence of the bacteria was analyzed with a scanning electron microscope. The quantitative data were related to the implant surface roughness (Pa-value) as measured by confocal laser scanning microscopy. RESULTS: Bacterial adherence of S. aureus varied significantly (p = 0.0035) with the type of metallic implant. Pure tantalum presented with significantly (p < 0.05) lower S. aureus adhesion compared to titanium alloy, polished stainless steel, and tantalum-coated stainless steel. Furthermore, pure tantalum had a lower, though not significantly, adhesion than commercially pure titanium and grit-blasted stainless steel. Additionally, there was a significantly higher S. aureus adherence to titanium alloy than to commercially pure titanium (p = 0.014). S. epidermidis adherence was not significantly different among the tested materials. There was no statistically significant correlation between bacterial adherence and surface roughness of the tested implants. CONCLUSIONS: Pure tantalum presents with a lower or similar S. aureus and S. epidermidis adhesion when compared with commonly used materials in orthopedic implants. CLINICAL IMPLICATION: Because bacterial adhesion is an important predisposing factor in the development of clinical implant infection, tantalum may offer benefits as an adjunct or alternative material compared with current materials commonly used for orthopedic implants.     

Titanium particles activate toll‐like receptor 4 independently of lipid rafts in RAW264.7 murine macrophages

Adherent pathogen‐associated molecular patterns (PAMPs) act through toll‐like receptor 2 (TLR2) and TLR4 to increase the biological activity of orthopedic wear particles in cell culture and animal models of implant loosening. This study tested whether this is dependent on TLR association with lipid rafts as reported for the response to soluble TLR ligands. For this purpose, RAW264.7 murine macrophages were activated by exposure to titanium particles with adherent PAMPs, soluble lipopolysaccharide (LPS), soluble lipotecichoic acid (LTA), or heat‐killed bacteria that had been extensively washed to remove soluble PAMPs. Lipid rafts were isolated by two independent methods and the location of TLR4 and TLR2 was analyzed by Western blotting. The cognate TLRs associated with lipid rafts when the macrophages were activated with soluble LPS and LTA but not after stimulation with either titanium particles with adherent PAMPs or heat‐killed bacteria. The lipid raft disruptor, methyl‐β‐cyclodextrin, dose‐dependently inhibited TNF‐α release in response to LPS but had no affect on TNF‐α release in response to titanium particles with adherent PAMPs. We conclude, therefore, that titanium particles with adherent PAMPs and heat‐killed bacteria activate TLR2 and TLR4 in macrophages without inducing either TLR to associate with lipid rafts. These results have important implications for the mechanisms of orthopedic implant loosening as well the mechanisms for TLR activation in other inflammatory situations. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:211–217, 2011     

Interaction of a pyelonephritogenic Escherichia coli strain with the tissue components of the mouse urinary tract

Immediately following the introduction of cells of a pyelonephritogenic strain of Escherichia coli (expressing both GS and MS adhesins) into the mouse bladder, these cells were found to be randomly distributed on the mucosal surfaces of both the bladder and the kidney. After 24 hr. these adherent bacterial cells had proliferated to form nonrandomly distributed adherent microcolonies on both mucosal surfaces and large masses within the renal ducts. Large amounts of amorphous material, which we presume to be of both host and bacterial origin, was associated with the bacterial microcolonies on the infected tissues. The removal of urinary slime and tissue coat material by acid pretreatment increased both the degree and the apparent avidity of bacterial adhesion to the bladder mucosa. The adherent bacteria on the bladder mucosa were radically elongated, while those on the kidney mucosa usually retained their short rod-like morphological characteristics. These data suggest that pyelonephritogenic bacteria adhere to mucosal surfaces in both the bladder and kidney, and proliferate to form adherent microcolonies within which bacteria are associated with large amounts of amorphous extracellular material.     

Role of surface roughness of titanium versus hydroxyapatite on human bone marrow cells response

This study was designed to assess human bone marrow cell response and particularly cell adhesion, proliferation, and differentiation, when cultured in vitro on titanium alloy and hydroxyapatite with different values of surface roughness. A further aim was to compare the cell response on the two materials, currently used in spinal surgery. Cell adhesion was determined after 0.5, 2, 4, and 18 hours of incubation; proliferation after 8, 11, 14, and 16 days of culture; and differentiation was evaluated with the expression of alkaline phosphatase activity after 8 and 16 days of culture. This study showed that bone marrow cells grew faster on titanium alloy than on hydroxyapatite, although fewer cells attached to titanium, compared to those attached to hydroxyapatite. No statistically significant difference was observed as the expression of alkaline phosphatase activity on hydroxyapatite and titanium alloy of the same roughness. Cell adhesion, proliferation, and differentiation are dependent on surface roughness of the biomaterial, and all three increased as the roughness of titanium alloy increased. Conclusively, surface roughness of titanium and hydroxyapatite significantly influences bone marrow cell response, and therefore these biomaterials should be used with rough outer surface, if a significant cell response on them is desired. These advantages of titanium and hydroxyapatite theoretically seem to be of particular importance in the following situations: long fusions, lumbosacral fusion, revision surgery with poor bone bank, neuropathic scoliosis associated with few bone graft reserves, and adult patients with severe osteoporosis.     

Chemiluminescent Responses of Macrophages Exposed to Biomaterials: A Biocompatibility Screening Test

Chemiluminescence (CL) response of alveolar macrophages (AM) exposed to either phagocytosable or nonphagocytosable particles of polymethylmethacrylate (PMMA) or titanium in vitro was monitored. The results were correlated to known in vivo bone tissue compatibility of the materials. Morever, the quantity and morphology of adherent AM on titanium and PMMA disks were assessed using scanning electron microscopy (SEM). Our results with PMMA indicated that a high CL response to phagocytosable as well as nonphagocytosable particles of the biomaterial in question, correlated with poor bone tissue compatibility. By contrast, a low CL response to phagocytosable particles and no response to nonphagocytosable particles correlated with a satisfactory bone tissue compatibility, as in the case of titanium. SEM showed 48% more AM adherent to PMMA than titanium per unit area. Also, the percentage of cells with membrane damage was three times higher on PMMA than on titanium. It appears that the CL response of AM exposed to biomaterials in vitro thus can be used as an easy to do screening test for potential orthopaedic implant materials before in vivo tests are considered     

Inhibition of the PI3K-Akt signaling pathway reduces tumor necrosis factor-alpha production in response to titanium particles in vitro

BACKGROUND: Wear debris contributes to implant loosening after total joint arthroplasty, and few advances have been made in our ability to inhibit the biological response to wear particles. Bacterial endotoxins augment the effects of wear particles in vitro and in vivo. The cytokine, tumor necrosis factor-alpha (TNF-alpha), is produced by macrophages in response to bacterial endotoxins and wear particles, and it increases osteoclast activity resulting in bone resorption and implant loosening. The phosphoinositol-3-kinase (PI3K)-Akt intracellular signal transduction pathway contributes to cytokine production in response to soluble endotoxin. We investigated the role of the PI3K-Akt pathway in the production of TNF-alpha in response to wear particles with adherent endotoxin and so-called endotoxin-free wear particles. METHODS: Cultured RAW264.7 murine macrophages were incubated with titanium particles with adherent endotoxin or with endotoxin-free titanium particles in the presence and absence of specific inhibitors of PI3K (LY294002) or Akt (SH-5). Akt activation was assessed with use of Western blot. TNF-alpha production was measured with use of enzyme-linked immunosorbent assay. Cytotoxicity was determined by measuring lactic dehydrogenase release. RESULTS: Titanium particles with adherent endotoxin increased Akt activation, whereas endotoxin-free titanium particles did not. The PI3K inhibitor reduced TNF-alpha production by 70% in response to titanium with adherent endotoxin without increasing cytotoxicity. Similarly, the Akt inhibitor reduced TNF-alpha production by 83% in response to titanium particles with adherent endotoxin without increasing cytotoxicity. High concentrations of endotoxin-free titanium particles resulted in a small delayed increase in TNF-alpha production that was completely blocked by the PI3K inhibitor. CONCLUSIONS: Inhibition of the PI3K-Akt pathway reduces macrophage TNF-alpha production in response to titanium particles with adherent endotoxin and endotoxin-free particles in vitro.     

Chemiluminescent responses of macrophages exposed to biomaterials: a biocompatibility screening test

Chemiluminescence (CL) response of alveolar macrophages (AM) exposed to either phagocytosable or nonphagocytosable particles of polymethylmethacrylate (PMMA) or titanium in vitro was monitored. The results were correlated to known in vivo bone tissue compatibility of the materials. Moreover, the quantity and morphology of adherent AM on titanium and PMMA disks were assessed using scanning electron microscopy (SEM). Our results with PMMA indicated that a high CL response to phagocytosable as well as nonphagocytosable particles of the biomaterial in question, correlated with poor bone tissue compatibility. By contrast, a low CL response to phagocytosable particles and no response to nonphagocytosable particles correlated with a satisfactory bone tissue compatibility, as in the case of titanium. SEM showed 48% more AM adherent to PMMA than titanium per unit area. Also, the percentage of cells with membrane damage was three times higher on PMMA than on titanium. It appears that the CL response of AM exposed to biomaterials in vitro thus can be used as an easy to do screening test for potential orthopaedic implant materials before in vivo tests are considered.     

Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro

Background and purpose Low-virulence implant infections are characterized by bacterial colonization of the implant with subsequent biofilm formation. In these cases, soft tissue biopsies often prove to be culture negative. Consequently, detachment of the causative adherent bacteria is crucial for correct microbiological diagnosis. Using an in vitro model, we compared 4 methods of biofilm sampling from metal surfaces.Methods Discs of titanium and steel were incubated in the presence of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Propionibacterium acnes in Mueller Hinton broth. Non-adherent bacteria were removed by repeated rinsing of the discs. 10 parallels of each disc were subjected to 1 of 4 methods for bacterial recovery: (A) sonication of the discs, (B) scraping of the discs using surgical blades followed by streaking of the blades onto agar plates, (C) scraping of the discs followed by vortex mixing of the surgical blades, and (D) scraping of the discs followed by sonication of the surgical blades. Quantitative bacterial cultures were performed for each sampling method.Results With the exception of S. epidermidis on steel, sonication efficiently and reliably dislodged biofilm bacteria. The scraping methods employed did not detach bacteria embedded in biofilm.Interpretation Scraping of metal surfaces is not an adequate method for sampling of biofilm bacteria in vitro.     

Bacterial adherence to surgical sutures. A possible factor in suture induced infection.

Surgical sutures are known to potentiate the development of wound infection. The purpose of this study was to investigate whether the capability of bacteria to adhere to various types of sutures has a significant effect on their ability to cause infections. Bacterial adherence to sutures was quantitatively measured using radiolabeled bacteria. In vitro adherence assays revealed remarkable variations in the affinity of bacteria to the various sutures: nylon bound the least bacteria while bacterial adherence to braided sutures (silk, Ti-cron, Dexon) was five to eight folds higher. The degree of infection obtained in mice in the presence of different sutures nicely correlated with their adherence properties. The different removal rate of adherent bacteria (glutaraldehyde-fixed) from various sutures by the tissue factors in mice supports the hypothesis that bacterial adherence to suture materials plays a significant role in the induction of surgical infection. Our observation points out at the need for careful suture selection in contaminated wounds. The adherence properties of sutures should be considered in any future surgical suture design.     

Electron microscopic study of an infected Foley catheter

A scanning and transmission electron microscopic study was made of material from the surface of a Foley catheter removed from an 87-year-old woman with antibiotic-resistant bacteriuria. A thick, adherent bacterial biofilm was found. The sessile adherent bacteria were surrounded by an extensive exopolysaccharide glycocalyx that appears to be fundamental in the pathogenesis of catheter-associated urinary tract infections and their resistance to systemic antibiotic therapy.     

Pull-out strength and bone tissue reaction of plasma-sprayed hydroxyapatite coatings with different thicknesses or substrates]

Synthetic hydroxyapatite has been reported to have good histocompatibility, while metal to have good mechanical properties in vivo. This author coated hydroxyapatite on SUS316L stainless steel and titanium rods, using plasma-spraying techniques. Then they were implanted into tubular bones of five dogs. The pull-out strength of hydroxyapatite-coated metals was found to be stronger than that of non-coated metals and increased with passage of the time. After four weeks postoperatively, the coating layer fractured between hydroxyapatite and the metal by the pull-out test. A numerous new bone in contact with hydroxyapatite was observed. Fifty microns is found to be an ideal thickness of the coating layer. The pull-out strength of hydroxyapatite-coated titanium was higher, comparing to that of hydroxyapatite-coated SUS316L stainless steel. These results suggested that perovskite and rutile existed at the interface between hydroxyapatite and titanium after plasma-spraying and made hydroxyapatite to bond tightly to the titanium. Synthetic perovskite showed no pathological reactions to canine bone tissue.     

In vitro quantitative adherence of bacteria to intravascular catheters

Adapting standard techniques, a simple in vitro system was devised to compare quantitative bacterial adherence to iv catheters of different compositions. Upon brief immersion of catheters in suspensions of Staphylococcus aureus, coagulase-negative staphylococci, and Escherichia coli, organisms adhered to catheter surfaces. After overnight growth in broth, organisms remained adherent and formed colonies, as shown by light and scanning electron microscopy. In addition, quantitative adherence using a blood agar roll technique, expressed as bacteria per square centimeter of catheter surface area per 10(6) colonies per milliliter inoculum, was calculated. Adherence was greater on polyvinylchloride (PVC) catheters (geometric mean 342) than on Teflon catheters (geometric mean 49.6) for coagulase-negative staphylococci (P less than 0.005). Also, the number of coagulase-negative staphylococci adherent to PVC catheters was significantly greater than for E. coli (geometric mean 70.6) at analogous inocula (P less than 0.02). Differences in bacterial adherence to the surface of iv devices may be important in the pathogenesis of catheter-associated infections. This in vitro method could prove useful in testing bacterial adherence properties of newly developed catheter materials, and allow development of catheters less prone to be associated with bacterial adherence and catheter-related infections.     

Experimental study of osteogenic activity of sintered hydroxyapatite--on the relationship of sintering temperature and pore size

In order to determine the most appropriate properties for hydroxyapatite, four kinds of sintered hydroxyapatite (50-250 microns & 900 degrees C, 50-250 microns & 1,100 degrees C, 300-600 microns & 1,100 degrees C, and dense & 1,100 degrees C, pore size and sintering temperature, respectively) were implanted in the animals and studied histologically. In addition, the responses at different implantation sites were compared. The results are 1) hydroxyapatite sintered at 900 degrees C temperature showed higher osteogenic activity than that sintered at 1,100 degrees C in the early period. However, this difference disappeared with time, and at 26 weeks, there was no significant difference between the two groups. 2) hydroxyapatite with a 300-600 microns pore size demonstrated osteogenic activity superior to that with a 50-250 microns pore size. 3) implantation at the epiphysis produced higher osteogenic activity in the early period, compared to implantations at the metaphysis or diaphysis. This difference in bone ingrowth had disappeared at the 26-week.     

Improved Detection of Biofilm-formative Bacteria by Vortexing and Sonication: A Pilot Study

Bacteria such as staphylococci commonly encountered in orthopaedic infections form biofilms and adhere to bone implants and cements. Various methods to disrupt the biofilm and enhance bacterial detection have been reported. We will describe the effectiveness of vortexing and sonication to improve the detection of biofilm-formative bacteria from polymethylmethacrylate by conventional quantitative bacterial culture and real-time quantitative PCR. We used a single biofilm-formative Staphylococcus aureus strain and 20 polymethylmethacrylate coupons as an in vitro biofilm model; four coupons were used for each of two control groups or three experimental sonication times (1, 5, and 30 minutes). Vortexing the cement without sonication increased the yield of adherent bacteria to a considerable extent. The combination of vortexing and sonication further enhanced the yield regardless of the duration of sonication. Quantitative conventional cultures correlated with quantitative PCR assay. The combination of vortexing and sonication to disrupt the bacterial biofilm followed by quantitative PCR and/or culture seems to be a sensitive method for detecting bacteria adherent to bone cement. Each author certifies that he or she has no commercial associations (eg, consultancies, stock ownership, equity interest, patent/licensing arrangements, etc) that might pose a conflict of interest in connection with the submitted article.     

In vivo resistance to bacterial biofilm formation on tympanostomy tubes as a function of tube material.

Adherent bacterial biofilms have been implicated in the irreversible contamination of implanted medical devices. We evaluated the resistance of various tympanostomy (pressure equalization [PE]) tube materials to biofilm formation using an in vivo model. PE tubes of silicone, silver oxide-impregnated silicone, fluoroplastic, silver oxide-impregnated fluoroplastic, and ion-bombarded silicone were inserted into the tympanic membranes of 18 Hartley guinea pigs. Staphylococcus aureus was then inoculated into the middle ears. An additional 8 guinea pigs were used as controls; the PE tubes were inserted without middle ear inoculation. All PE tubes were removed on day 10 and analyzed for bacterial contamination using culture, immunofluorescence, and scanning electron microscopy (SEM). All infected ears developed otitis media with otorrhea, but none of the animal control ears drained. Fluorescence imaging of the animal control tubes showed large cellular components consistent with inflammation. The infected tubes showed heavy DNA fluorescence consistent with bacteria and inflammatory cells. All animal control tubes except the ion-bombarded silicone tubes showed adherent inflammatory film on SEM. Also, all tubes placed in infected ears except the ion-bombarded silicone tubes showed adherent bacterial and inflammatory films on SEM. Nonadherent surface properties such as the ion-bombarded silicone may be helpful in preventing chronic PE tube contamination.