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.     

Opening plugged tympanostomy tubes: effect of biofilm formation.

OBJECTIVE: To determine if the presence of a bacterial biofilm impacts the rate of clearing of mucoid plugs from tympanostomy tubes (TTs). STUDY DESIGN AND SETTING: Ex vivo model. Stainless steel Reuter Bobbin TTs (n = 18) were placed in growth medium with Pseudomonas aeruginosa and Streptococcus pneumoniae for 12 days to promote biofilm formation. Tympanostomy tubes (n = 18) placed in growth medium, without bacteria, for 12 days served as controls. Biofilm formation was assessed by scanning electron microscopy. All TTs were filled with middle ear mucus and allowed to dry, thereby forming a plug. TTs were placed in a model ear chamber, covered with ofloxacin otic solution, and the time to clear each plug was recorded. RESULTS: Biofilm formation was consistently encountered on TTs exposed to bacteria but in no TTs in the control group. There was a significant effect of the biofilm on plug clearance, favoring TTs without a biofilm (P = 0.0333). Although there was no significant difference in the proportion of unplugged TTs (P = 0.264), TTs with a biofilm did not clear plugs as rapidly as TTs without a biofilm (P = 0.0416). CONCLUSIONS: The presence of a biofilm may slow the time to clear mucoid TT plugs, but it does not seem to affect the overall proportion of TTs that are unplugged.     

In vitro testing of tympanostomy tube occlusion

ObjectiveTympanostomy tubes (TTs) are commonly rendered nonfunctional by mucus plug formation. The purpose of this study was to determine whether an in vitro model could be developed to assess TT plug formation with results consistent with human trials.Study DesignAn ear chamber was designed to mimic middle ear air and mucus flow conditions in post-TT otorrhea. TT occlusion was tested and correlated to published in vivo results.MethodsTTs that had previously been studied in vivo (Goode “T” and Reuter Bobbin collar buttons) were placed in the model chamber. Pooled, homogenized human middle ear mucus and an analog, egg white, were delivered at 80 μL per hour through the TTs. An air bolus was delivered every two minutes to simulate swallowing. Chamber pressure was monitored over 2.5 hours. Occlusion was determined by a pressure peak and visual confirmation.ResultsObstruction was found in 60 percent of the Reuter Bobbin and 40 percent of the Goode TTs using the mucus analog. These results are similar to those reported from previous in vivo studies. No plugging was reported for either TT using homogenized human ear mucus.ConclusionsThe in vitro TT chamber simulates the in vivo environment and yields results consistent with in vivo observations. This model system may allow for rapid prototyping and evaluation of new TTs that may be less vulnerable to occlusion.     

Microbiology of acute otitis media with tympanostomy tubes.

OBJECTIVE: The objective was to determine the types of organisms which cause acute otitis media with a tympanostomy tube and to ascertain their frequency distribution. STUDY DESIGN AND SETTING: Prospective, randomized, multi-institutional clinical trials. Both private and academic sites were included. RESULTS: 1309 isolates were recovered from 956 draining ears. Streptococcus pneumonia was recovered from 17%, Staphylococcus aureus from 13%, H flu from 18% and Pseudomonas aeruginosa from 12%. Fungal organisms were recovered from 5% of total isolates and 4% from single isolates. CONCLUSIONS: AOMT is microbiologically different than AOM with an intact TM. There is no evidence that resistance develops as result of topical treatment. SIGNIFICANCE: The study demonstrates that AOMT is frequently caused by organisms not susceptible to oral antibiotics approved for children, but which are sensitive to topical ear drops.     

Experimental and clinical experience of albumin coating of tympanostomy tubes.

OBJECTIVE: Otorrhea and tube occlusion are typical problems with tympanostomy tubes (TT). The purpose of this study was to test glue protein, fibronectin adhesion on albumin-coated and uncoated TT surfaces and to show the effect of this method on TT sequelae in vivo. STUDY DESIGN AND SETTINGS: Fibronectin binding on TT surface was tested in two in vitro experiments. Thereafter 170 patients were randomized in a prospective clinical trial to test the effect of the method in vivo. The extruded TTs that could be collected from ears of some study patients were imaged with scanning electron microscopy (SEM). RESULTS: Marked binding inhibition of fibronectin on albumin-coated TTs was found. Fewer tube sequelae were found in ears with albumin-coated TTs. SEM revealed thick crusts on uncoated tube surface. CONCLUSION: Albumin coating of TTs can reduce unwanted adhesion on the tube surface in vitro and tube occlusions in vivo. SIGNIFICANCE: TT sequelae can be reduced by surface coating with albumin.     

Microbial adhesion and biofilm formation on ureteral stents in vitro and in vivo.

Thirty ureteral stents, inserted for 5 to 128 days following extracorporeal shock wave lithotripsy, were examined for the presence of bacterial biofilms. Of these, 90% had adherent pathogens (44% mixed organisms) on the stents, 45% of which were present in low numbers (10(1)-10(2) per 1 cm3 section) and 55% were in small and large microcolony biofilms (> 2 x 10(2)-10(7)). The organisms were recovered from the stents even though urine culture was only positive in 27% of patients. Of the organisms isolated, 77% were Gram positive cocci, 15% Gram negative rods and 8% Candida. No blockage of the stents occurred. All of the patients had received antimicrobial therapy post-insertion, and in 15 cases biofilms were found while on treatment. None of the patients received therapy for urinary tract infections while the stent remained in place. In vitro experiments demonstrated the ability of Escherichia coli, Proteus mirabilis, Staphylococcus epidermidis and Enterococcus faecalis uropathogens to adhere and form biofilms on ureteral stents within 24 hours. Clearly, bacterial biofilms do occur on ureteral stents and urinary culture may not detect their presence. The high recovery rate of Gram positive organisms may indicate a preferential adhesion to the biomaterial surface. The findings also indicate that unlike biofilm formation on many other prosthetic implants, colonization with Gram positive organisms on ureteral stents does not necessarily coincide with the development symptomatic infection.     

Opening plugged tympanostomy tubes: effect of inner diameter and shaft length.

OBJECTIVE: To determine whether tympanostomy tube (TT) inner diameter or shaft length impacts the rate of mucoid plug clearance. STUDY DESIGN AND SETTING: Ex vivo model. Silicone TTs with different inner-diameters (ID) and shaft-length (SL) pairings (1.14 mm ID x 12 mm SL versus 1.14 mm ID x 1 mm SL; 1.14 mm ID x 4.8 mm SL versus 1.32 mm ID x 4.8 mm SL) were plugged with middle-ear mucus (n = 15 per group) and placed in a model ear chamber. Ofloxacin otic solution was instilled into the chamber to cover the plugged TT, and the time to clearance of each plug was recorded. RESULTS: TTs with larger IDs ( P = 0.019) and greater SLs ( P = 0.033) cleared plugs more rapidly. However, the difference in the percentage of tubes that unplugged was not significant ( P = 0.151). CONCLUSIONS: Rate of ex vivo TT plug clearance may be altered by changing TT ID and SL.     

防治生物膜细菌活性成分的研究进展

近年来抗生物膜细菌活性成分的研究主要集中在化学合成物、天然产物(包括植物中的活性成分和海洋生物活性成分)、生物大分子活性成分等方面,各类别中都具有一定的抑制和(或)杀灭生物膜作用,然而大多数抗生物膜活性成分不足以完全抑制或杀灭生物膜,缺乏广谱抗生物膜制剂,需要对生物膜形成机制认识的不断深入及现代生物医药技术的不断进步。该文对防治生物膜细菌活性成分的研究进行综述,以为下一步研究临床上生物膜细菌形成感染的防治提供依据。     

Effects of biofilm formation on haemodialysis monitor disinfection.

BACKGROUND: Biofilms are composed of communities of micro-organisms adhering to essentially any surface. We evaluated whether biofilm formation in the hydraulic circuit of a purposely contaminated haemodialysis monitor would modify the efficacy of different disinfection modalities against bacteria and endotoxin concentrations. METHODS: A water-borne Pseudomonas aeruginosa (109) suspension was recirculated for 1 h and was left standing for 72 h (stationary phase) in the hydraulic circuit of the monitor. The monitor was then washed and disinfected by different physical (heat, 85 degrees C) or chemical (hypochlorite or peracetic acid) disinfection modalities (protocol A). In protocol B, the bacterial suspension was also recirculated for 1 h, but the monitor was then immediately washed and disinfected by different chemical disinfection modalities (hypochlorite or peracetic acid). RESULTS: Biofilm formation was revealed by scanning and confocal laser electron microscopy after the stationary phase (protocol A), but was absent when the monitor was immediately washed and disinfected (protocol B). In the presence of biofilm (protocol A), heat in association with citric acid was the most effective modality for reducing both colony forming units and endotoxin concentrations, whereas heat by itself was the least effective method of disinfection. Dwelling (60 h) with diluted peracetic acid completely prevented the formation of biofilm. In the absence of biofilm (protocol B), chemical disinfection proved to be effective against both colony forming units and endotoxin concentrations. CONCLUSIONS: We found that biofilm formation may markedly reduce the efficacy of presently available disinfection modalities. Therefore, different disinfection modalities and the combined action of descaling (by citric acid) and disinfection (physical/chemical agents) should be used periodically in haemodialysis monitors. In addition, dwelling with diluted peracetic acid should be adopted whenever monitors are not in use.     

Prophylactic antibiotics prevent bacterial biofilm graft infection.

Bacterial biofilm graft infection is due to prostheses colonization by Staphylococcus epidermidis, a pathogen frequently recovered from perigraft tissues of man during vascular procedures despite the use of asepsis and prophylactic antibiotics. The effect of preoperative intraperitoneal cefazolin, administered at a standard (15 or 30 mg/kg) and high (120 mg/kg) dose, on the prevention of bacterial biofilm infection was studied in a rat model. Seventy-four Dacron grafts, colonized in vitro with S. epidermidis to produce an adherent biofilm (3.19 +/- 0.71 x 10(7) colony-forming units/cm2 graft), were implanted in the dorsal subcutaneous tissue at 0.5, 2, and 4 hr after antibiotic administration. The study strain was a slime-producing clinical isolate with minimum inhibitory concentration (MIC) of 15-30 micrograms/ml to cefazolin. Subcutaneous tissue antibiotic levels were determined at each time interval. One week after implantation, the concentration of bacteria in the surface biofilm by quantitative agar culture was significantly decreased (P less than 0.05) only for grafts implanted when antibiotic tissue levels were greater than or equal to the MIC of the study strain. The result of no growth by biofilm broth culture was significantly achieved (P less than 0.01) only for grafts implanted 0.5 hr after high dose cefazolin, in which the tissue antibiotic level was above the MIC of the study strain. Antibiotics can markedly reduce the bacteria concentration of a prosthetic surface biofilm. The effectiveness of prophylactic antibiotics on the prevention of graft infection is dependent upon maintaining an adequate antibiotic level in the perigraft tissues for the duration of the procedure.     

Electron microscopic analysis of biofilm on endotracheal tubes removed from intubated neonates.

OBJECTIVES: To determine if the phenomenon of biofilm accumulation and associated microbial colonization occurs on the surface of endotracheal tubes in the region of the subglottis in neonates. METHODS: Endotracheal tubes removed from 9 consecutive neonatal patients intubated for more than 12 hours were processed (range, 13 hours to 8 days). A sterile control tube was also processed. For each, the portion of the endotracheal tube that had been in contact with the subglottis was determined using a previously published nomogram. A 1-cm-long cross-sectional segment of the endotracheal tube corresponding to the level of the subglottis was divided into 2 portions for both electron microscopy and aerobic/anaerobic cultures. RESULTS: Two of 9 (22%) luminal surface cultures grew Staphylococcus species, 1 (11%) grew normal flora, and 6 (66%) had no growth. Three of 9 (33%) outer-surface cultures grew Staphylococcus species, 1 (11%) had gram-negative rods on staining but a sterile culture, and one enterococcal contaminant was found. Electron microscopy revealed that 8 of 9 inner lumen surfaces harbored bacteria and biofilm formation. All outer lumen surfaces had biofilm formation; 6 of 9 had bacterial colonization. There was no obvious difference in the appearance of the inner and outer tube surface accretions. No time-dependent differences were noted except of the longest indwelling tube (8 days). CONCLUSION: This study demonstrates for the first time the presence of biofilm on the outer surface of neonatal endotracheal tubes. The data suggest that the presence of bacteria and/or biofilm does not correlate with other traditional indicators of microbial colonization.     

Medical decision analysis: indications for tympanostomy tubes in RAOM by age at first episode.

OBJECTIVE: To compare utility estimates between tympanostomy tubes (TT) and short-courses of antibiotics in children with recurrent acute otitis media (RAOM) stratified by age at first episode. STUDY DESIGN AND SETTING: Formal decision analysis. RESULTS: The model recommended TT sooner in children with a history of a first episode of AOM occurring early in life. In children over 12 months old at onset, TT were recommended with seven episodes in 24 months, five episodes in 12 months, and three episodes in six months. In children under six months old at onset, TT were recommended with three episodes in 24 months and two episodes in a six-month or 12-month time span. CONCLUSIONS: Earlier TT may be indicated in children who developed a first episode of AOM at a very young age because of the higher risk of AOM recurrence. SIGNIFICANCE: This study is the first formal decision analysis to compare tympanostomy tubes and short-courses of antibiotics stratified by age at onset of the first AOM episode.     

In vitro release of vancomycin from vancomycin-loaded blood coated demineralised bone

In vitro and in vivo studies have demonstrated the possibility that cancellous bone could be used as a carrier of antibiotics for local delivery. However, the release of antibiotics from the loaded cancellous bone is too rapid and uncertain. We hypothesised that demineralisation of cancellous bone would increase the amount of antibiotic adsorbed, and coating of the freeze-dried antibiotic-impregnated cancellous bone with bio-compatible material would prolong antibiotic release. Bovine cancellous bone blocks of equal size were demineralised using a 0.5 N HCl solution and loaded with vancomycin solution under vacuum. The loaded bone blocks were then freeze-dried. To obtain a bio-compatible coating, the vancomycin-impregnated bone blocks were soaked in fresh human venous blood for 3 h. The release of impregnated antibiotic from the bone blocks was evaluated in phosphate-buffered saline and foetal bovine serum. It was found that significantly larger amounts of vancomycin were adsorbed into the demineralised bone blocks than into the un-demineralised blocks. The blood coating was found to increase the duration of vancomycin release from the blocks. With demineralisation and blood coating, the blocks eluted vancomycin higher than therapeutic concentration for a 5-week period.     

An in vitro biofilm model to examine the effect of antibiotic ointments on biofilms produced by burn wound bacterial isolates

Purpose

Topical treatment of burn wounds is essential as reduced blood supply in the burned tissues restricts the effect of systemic antibiotics. On the burn surface, microorganisms exist within a complex structure termed a biofilm, which enhances bacterial resistance to antimicrobial agents significantly. Since bacteria differ in their ability to develop biofilms, the susceptibility of these biofilms to topically applied antibiotics varies, making it essential to identify which topical antibiotics efficiently disrupt or prevent biofilms produced by these pathogens. Yet, a simple in vitro assay to compare the susceptibility of biofilms produced by burn wound isolates to different topical antibiotics has not been reported.

Methods

Biofilms were developed by inoculating cellulose disks on agar plates with burn wound isolates and incubating for 24 h. The biofilms were then covered for 24 h with untreated gauze or gauze coated with antibiotic ointment and remaining microorganisms were quantified and visualized microscopically.

Results

Mupirocin and triple antibiotic ointments significantly reduced biofilms produced by the Staphylococcus aureus and Pseudomonas aeruginosa burn wound isolates tested, as did gentamicin ointment, with the exception of one P. aeruginosa clinical isolate.

Conclusions

The described assay is a practical and reproducible approach to identify topical antibiotics most effective in eliminating biofilms produced by burn wound isolates.     

A study on the ability of quaternary ammonium groups attached to a polyurethane foam wound dressing to inhibit bacterial attachment and biofilm formation

Bacterial infection of acute and chronic wounds impedes wound healing significantly. Part of this impediment is the ability of bacterial pathogens to grow in wound dressings. In this study, we examined the effectiveness of a polyurethane (PU) foam wound dressings coated with poly diallyl‐dimethylammonium chloride (pDADMAC‐PU) to inhibit the growth and biofilm development by three main wound pathogens, Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, within the wound dressing. pDADMAC‐PU inhibited the growth of all three pathogens. Time‐kill curves were conducted both with and without serum to determine the killing kinetic of pDADMAC‐PU. pDADMAC‐PU killed S. aureus, A. baumannii, and P. aeruginosa. The effect of pDADMAC‐PU on biofilm development was analyzed quantitatively and qualitatively. Quantitative analysis, colony‐forming unit assay, revealed that pDADMAC‐PU dressing produced more than eight log reduction in biofilm formation by each pathogen. Visualization of the biofilms by either confocal laser scanning microscopy or scanning electron microscopy confirmed these findings. In addition, it was found that the pDADMAC‐PU‐treated foam totally inhibited migration of bacteria through the foam for all three bacterial strains. These results suggest that pDADMAC‐PU is an effective wound dressing that inhibits the growth of wound pathogens both within the wound and in the wound dressing.     

Endotracheal tubes coated with antiseptics decrease bacterial colonization of the ventilator circuits, lungs, and endotracheal tube

BACKGROUND: Formation of a bacterial biofilm within the endotracheal tube (ETT) after tracheal intubation is rapid and represents a ready source of lung bacterial colonization. The authors investigated bacterial colonization of the ventilator circuit, the ETT, and the lungs when the ETT was coated with silver-sulfadiazine and chlorhexidine in polyurethane, using no bacterial/viral filter attached to the ETT. METHODS: Sixteen sheep were randomized into two groups. Eight sheep were intubated with a standard ETT (control group), and eight were intubated with a coated ETT (study group). Animals were mechanically ventilated for 24 h. At autopsy, the authors sampled the trachea, bronchi, lobar parenchyma, and ETT for quantitative bacterial cultures. Qualitative bacterial cultures were obtained from the filter, humidifier, inspiratory and expiratory lines, and water trap. ETTs were analyzed with light microscopy, scanning electron microscopy, and laser scanning confocal microscopy. RESULTS: In the control group, all eight ETTs were heavily colonized (10(5)-10(8) colony-forming units [cfu]/g), forming a thick biofilm. The ventilator circuit was always colonized. Pathogenic bacteria colonized the trachea and the lungs in five of eight sheep (up to 10(9) cfu/g). In the study group, seven of eight ETTs and their ventilator circuits showed no growth, with absence of a biofilm; one ETT and the respective ventilator circuit showed low bacterial growth (10(3)-10(4) cfu/g). The trachea was colonized in three sheep, although lungs and bronchi showed no bacterial growth, except for one bronchus in one sheep. CONCLUSIONS: Coated ETTs induced a nonsignificant reduction of the tracheal colonization, eliminated (seven of eight) or reduced (one of eight) bacterial colonization of the ETT and ventilator circuits, and prevented lung bacterial colonization.     

Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces.

BACKGROUND: Microbial biofilms form on central venous catheters and may be associated with systemic infections as well as decreased dialysis efficiency due to catheter thrombosis. The most widely used anticoagulant catheter lock solution in the US is sodium heparin. We have previously shown that sodium heparin in clinically relevant concentrations enhances Staphylococcus aureus biofilm formation. In the present study, we examine the effect of several alternative catheter lock solutions on in vitro biofilm formation by laboratory and clinical isolates of S. aureus and coagulase-negative staphylococci (CNS). METHODS: Lepirudin, low molecular weight heparin, tissue plasminogen activator, sodium citrate, sodium citrate with gentamicin and sodium ethylene diamine tetra-acetic acid (EDTA) were assessed for their effect on biofilm formation on polystyrene, polyurethane and silicon elastomer. RESULTS: Sodium citrate at concentrations above 0.5% efficiently inhibits biofilm formation and cell growth of S. aureus and Staphylococcus epidermidis. Subinhibitory concentrations of sodium citrate significantly stimulate biofilm formation in most tested S. aureus strains, but not in CNS strains. Sodium EDTA was effective in prevention of biofilm formation as was a combination of sodium citrate and gentamicin. Low molecular weight heparin stimulated biofilm formation of S. aureus, while lepirudin and tissue plasminogen activator had little effect on S. aureus biofilm formation. CONCLUSIONS: This in vitro study demonstrates that heparin alternatives, sodium citrate and sodium EDTA, can prevent the formation of S. aureus biofilms, suggesting that they may reduce the risk of biofilm-associated complications in indwelling catheters. This finding suggests a biological mechanism for the observed improvement in catheter-related outcomes in recent clinical comparisons of heparin and trisodium citrate as catheter locking solutions. A novel and potential clinically relevant finding of the present study is the observation that citrate at low levels strongly stimulates biofilm formation by S. aureus.     

EPA covalently bound to smooth titanium surfaces decreases viability and biofilm formation of Staphylococcus epidermidis in vitro

Colonization of implant surfaces with bacteria should ideally be prevented right from implantation, as bacteria attaching to the surface will form a biofilm, being then well protected against antibiotic treatment. Therefore, implant coatings should combine antibacterial properties with biocompatibility towards their host tissue. We tested a UV‐induced covalent coating procedure with eicosapentaenoic acid (EPA) for smooth titanium (Ti) surfaces for its ability to prevent attachment and proliferation of Staphylococcus epidermidis and to allow mineralization of MC3T3‐E1 osteoblasts. Bacterial initial attachment was highest for EPA‐coated surfaces, but was reduced by vigorous washing, possibly due to low adhesive strength on those surfaces. We found an increase in the ratio of dead bacteria and in overall biofilm after 16 h on Ti surfaces with covalently bound EPA compared to Ti. The UV‐induced EPA coating did not impair the ability of MC3T3‐E1 preosteoblasts to mineralize, while a reduction in mineralization could be found for UV‐irradiated Ti surfaces and UV‐irradiated surfaces washed with ethanol compared to Ti. Although in vivo studies are needed to evaluate the clinical significance, our results indicate that covalent coating of Ti surfaces with EPA by UV irradiation decreases the survival of S. epidermidis and maintains the mineralization ability of osteoblasts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1384–1390, 2012