Eradication of endotracheal tube biofilm by nebulised gentamicin.

Objective: To compare the efficacy of gentamicin, nebulised via the endotracheal tube (ET), with that of parenteral cefotaxime or parenteral cefuroxime in preventing the formation of ET biofilm.

Setting: General intensive care units in two university teaching hospitals.

Design: The microbiology of ET biofilm from 36 ICU patients eligible to receive antibiotic prophylaxis was examined. Peak and trough tracheal concentrations of gentamicin, cefotaxime or cefuroxime were measured in each patient group, on the 2nd day of intubation.

Patients: Twelve patients received gentamicin (80 mg) nebulised in 4 ml normal saline every 8 h, 12 cefotaxime (1 g, 12 hourly) and 12 cefuroxime (750 mg, 8 hourly). Prophylaxis was continued for the duration of intubation.

Measurements and results: Samples of tracheal secretions were taken on the 2nd day of ventilation for determination of antibiotic concentrations. Following extubation, ETs were examined for the presence of biofilm. Pathogens considered to be common aetiological agents for VAP included Staphylococcus aureus, enterococci, Enterobacteriaceae and pseudomonads. While microbial biofilm was found on all ETs from the cephalosporin group, microbial biofilm of these micro-organisms was found on 7 of the 12 ET tubes from patients receiving cefotaxime [S. aureus (4), pseudomonads (1), Enterobacteriaceae (1), enterococcus (1)] and 8 of the 12 ET tubes from patients receiving cefuroxime [Enterobacteriaceae (6), P. aeruginosa (1) and enterococcus (1)]. While microbial biofilm was observed on five ETs from patients receiving nebulised gentamicin, none of these were from pathogens for ventilator-associated pneumonia (VAP). Tracheal concentrations of both cephalosporins were lower than those needed to inhibit the growth of pathogens recovered from ET tube biofilm. The median (and range) concentrations for cefotaxime were 0.90 (<0.23–1.31) mg/l and 0.28 (<0.23–0.58) mg/l for 2 h post-dose and trough samples, respectively. Two hours post-dose concentrations of cefuroxime (median and range) were 0.40 (0.34–0.83) mg/l, with trough concentrations of 0.35 (<0.22–0.47) mg/l. Tracheal concentrations (median and range) of gentamicin measured 1 h post-nebulisation were 790 (352–>1250) mg/l and then, before the next dose, were 436 (250–1000) mg/l.

Conclusion: Nebulised gentamicin attained high concentrations in the ET lumen and was more effective in preventing the formation of biofilm than either parenterally administered cephalosporin and therefore may be effective in preventing this complication of mechanical ventilation.

In vitro study of the efficacy of acrylic bone cement loaded with supplementary amounts of gentamicin: Effect on mechanical properties, antibiotic release, and biofilm formation

Background: Infection remains a severe complication following a total hip replacement. If infection is suspected when revision surgery is being performed, additional gentamicin is often added to the cement on an ad hoc basis in an attempt to reduce the risk of recurrent infection.

Methods and results: In this in vitro study, we determined the effect of incorporating additional gentamicin on the mechanical properties of cement. We also determined the degree of gentamicin release from cement, and also the extent to which biofilms of clinical Staphylococcus spp. isolates form on cement in vitro. When gentamicin was added to unloaded cement (1–4 g), there was a significant reduction in the mechanical performance of the loaded cements compared to unloaded cement. A significant increase in gentamicin release from the cement over 72 h was apparent, with the amount of gentamicin released increasing significantly with each additional 1 g of gentamicin added. When overt infection was modeled, the incorporation of additional gentamicin did result in an initial reduction in bacterial colonization, but this beneficial effect was no longer apparent by 72 h, with the clinical strains forming biofilms on the cements despite the release of high levels of gentamicin.

Interpretation: Our findings indicate that the addition of large amounts of gentamicin to cement is unlikely to eradicate bacteria present as a result of an overt infection of an existing implant, and could result in failure of the prosthetic joint because of a reduction in mechanical performance of the bone cement.

In vitro testing of chitosan in gentamicin-loaded bone cement No antimicrobial effect and reduced mechanical performance

Background and purpose Efforts to prevent infection of arthroplasties, including the use of antibiotic-loaded bone cement, are not always successful. We investigated whether the incorporation of chitosan in gentamicin-loaded bone cement increases antibiotic release, and prevents bacterial adherence and biofilm formation by clinical isolates of Staphylococcus spp. In addition, we performed mechanical and degradation tests.

Gentamicin-loaded nanoparticles show improved antimicrobial effects towards Pseudomonas aeruginosa infection

Gentamicin is an aminoglycoside antibiotic commonly used for treating Pseudomonas infections, but its use is limited by a relatively short half-life. In this investigation, developed a controlled-release gentamicin formulation using poly(lactide-co-glycolide) (PLGA) nanoparticles. We demonstrate that entrapment of the hydrophilic drug into a hydrophobic PLGA polymer can be improved by increasing the pH of the formulation, reducing the hydrophilicity of the drug and thus enhancing entrapment, achieving levels of up to 22.4 µg/mg PLGA. Under standard incubation conditions, these particles exhibited controlled release of gentamicin for up to 16 days. These particles were tested against both planktonic and biofilm cultures of P. aeruginosa PA01 in vitro, as well as in a 96-hour peritoneal murine infection model. In this model, the particles elicited significantly improved antimicrobial effects as determined by lower plasma and peritoneal lavage colony-forming units and corresponding reductions of the surrogate inflammatory indicators interleukin-6 and myeloperoxidase compared to free drug administration by 96 hours. These data highlight that the controlled release of gentamicin may be applicable for treating Pseudomonas infections.

Construction of aminoglycoside-sensitive Burkholderia cenocepacia strains for use in studies of intracellular bacteria with the gentamicin protection assay

Burkholderia cenocepacia is a multidrug-resistant opportunistic pathogen that infects the airways of patients with cystic fibrosis (CF) and can survive intracellularly in macrophages and epithelial cells. The gentamicin protection assay, which relies on the poor ability of gentamicin or other aminoglycosides to permeate eukaryotic cell membranes, is traditionally employed to quantify intracellular bacteria. However, the high resistance of these bacteria to aminoglycosides hampers the use of the gentamicin protection assay to investigate intracellular infection by B. cenocepacia. Here, we report the construction of gentamicin-sensitive strains of B. cenocepacia carrying a deletion of the BCAL1674, BCAL1675, and BCAL1676 genes that form an operon encoding an AmrAB-OprA-like efflux pump. We show that bacteria carrying this deletion are hypersensitive to gentamicin and also delay phagolysosomal fusion upon infection of RAW 264.7 murine macrophages, as previously demonstrated for the parental strain. We also demonstrate for the first time that low concentrations of gentamicin can be used to effectively kill extracellular bacteria and reliably quantify the intracellular infection by B. cenocepacia, which can replicate in RAW 264.7 macrophages.

Biofilm formation by bacteria isolated from retrieved failed prosthetic hip implants in an in vitro model of hip arthroplasty antibiotic prophylaxis

Bacterial infection primarily with Staphylococcus spp. and Propionibacterium acnes remains a significant complication following total hip replacement. In this in vitro study, we investigated the efficacy of gentamicin loading of bone cement and pre- and postoperative administration of cefuroxime in the prevention of biofilm formation by clinical isolates. High and low initial inocula, representative of the number of bacteria that may be present at the operative site as a result of overt infection and skin contamination, respectively, were used. When a high initial inoculum was used, gentamicin loading of the cement did not prevent biofilm formation by the 10 Staphylococcus spp. and the 10 P. acnes isolates tested. Similarly, the use of cefuroxime in the fluid phase with gentamicin-loaded cement did not prevent biofilm formation by four Staphylococcus spp. and four P. acnes isolates tested. However, when a low bacterial inoculum was used, a combination of both gentamicin-loaded cement and cefuroxime prevented biofilm formation by these eight isolates. Our results indicate that this antibiotic combination may protect against infection after intra-operative challenge with bacteria present in low numbers as a result of contamination from the skin but would not protect against bacteria present in high numbers as a result of overt infection of an existing implant.

Impact of antibiotics on conjugational resistance gene transfer in Staphylococcus aureus in sewage.

Ohlsen K, Ternes T, Werner G, Wallner U, Löffler D, Ziebuhr W, Witte W, Hacker J. Institute for Molecular Biology of Infectious Diseases, The University of Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. knut.ohlsen@mail.uni-wuerzburg.de The growing rate of microbial pathogens becoming resistant to standard antibiotics is an important threat to public health. In order to assess the role of antibiotics in the environment on the spread of resistance factors, the impact of subinhibitory concentrations of antibiotics in sewage on gene transfer was investigated using conjugative gentamicin resistance (aacA-aphD) plasmids of Staphylococcus aureus. Furthermore, the concentration of antibiotics in hospital sewage was measured by high-performance liquid chromatography (HPLC)-electrospray tandem mass spectrometry. Several antibiotics were found to be present in sewage, e.g. ciprofloxacin up to 0.051 mgl(-1) and erythromycin up to 0.027 mgl(-1). Resistance plasmid transfer occurred both on solidified (dewatered) sewage and in liquid sewage in a bioreactor with a frequency of 1.1x10(-5)-5.0x10(-8). However, low-level concentrations of antibiotics measured in sewage are below concentrations that can increase plasmid transfer frequencies of gentamicin resistance plasmids of staphylococci.

Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis.

Rachid S, Ohlsen K, Witte W, Hacker J, Ziebuhr W. Institut für Molekulare Infektionsbiologie, Röntgenring 11, D-97070 Würzburg, Germany. Biofilm production is an important step in the pathogenesis of Staphylococcus epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin. A chromosomally encoded reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from Escherichia coli was constructed and used to investigate the influence of both environmental factors and subinhibitory concentrations of different antibiotics on ica expression in S. epidermidis. It was shown that S. epidermidis biofilm formation is induced by external stress (i.e., high temperature and osmolarity). Subinhibitory concentrations of tetracycline and the semisynthetic streptogramin antibiotic quinupristin-dalfopristin were found to enhance ica expression 9- to 11-fold, whereas penicillin, oxacillin, chloramphenicol, clindamycin, gentamicin, ofloxacin, vancomycin, and teicoplanin had no effect on ica expression. A weak (i.e., 2.5-fold) induction of ica expression was observed for subinhibitory concentrations of erythromycin. The results were confirmed by Northern blot analyses of ica transcription and quantitative analyses of biofilm formation in a colorimetric assay.

Incorporation of chitosan in acrylic bone cement: Effect on antibiotic release, bacterial biofilm formation and mechanical properties

Bacterial infection remains a significant problem following total joint replacement. Efforts to prevent recurrent implant infection, including the use of antibiotic-loaded bone cement for implant fixation at the time of revision surgery, are not always successful. In this in vitro study, we investigated whether the addition of chitosan to gentamicin-loaded Palacos® R bone cement increased antibiotic release and prevented bacterial adherence and biofilm formation by Staphylococcus spp. clinical isolates. Furthermore, mechanical tests were performed as a function of time post-polymerisation in pseudo-physiological conditions. The addition of chitosan to gentamicin-loaded Palacos® R bone cement significantly decreased gentamicin release and did not increase the efficacy of the bone cement at preventing bacterial colonisation and biofilm formation. Moreover, the mechanical performance of cement containing chitosan was significantly reduced after 28 days of saline degradation with the compressive and bending strengths not in compliance with the minimum requirements as stipulated by the ISO standard for PMMA bone cement. Therefore, incorporating chitosan into gentamicin-loaded Palacos® R bone cement for use in revision surgery has no clinical antimicrobial benefit and the detrimental effect on mechanical properties could adversely affect the longevity of the prosthetic joint.

Liquid marble formation using hydrophobic powders

This work aims to investigate and quantitatively measure “liquid marble” phenomena using hydrophobic powders (granules). The hydrophobic powders based on a copper substrate were prepared by a silver deposition technique of particle sizes 9 µm, 20 µm and 320 µm and of contact angle with water approaching 160°. The hydrophobic powder poly-methylmethacralate (PMMA) particle size 42 µm and contact angle of 120° was also used to determine the effect of powder density on liquid marble stability. The experimental investigations indicated that for successful formation of liquid marbles a number of variables in addition to hydrophobicity need to be considered, namely: powder density; powder particle size; powder shape; liquid marble formation technique. It was found that liquid marbles were formed using all four powders to varying extents, with a low powder particle size forming more stable liquid marbles. In a series of gravimetric tests, adhered powder mass on liquid marbles was found to be directly proportional to the water droplet surface area. A more complete coverage of the water drops were found with PMMA powder than the hydrophobic granules. Moreover, a further procedure was developed to increase the mechanical strength of the liquid marble, by polymerising methylmethacrylate (MMA) on the surface of a PMMA powder – liquid marble, with the aim of maintaining water within a more robust PMMA – liquid marble shell. This technique may prove to be a novel way of encapsulating drug compounds, such as gentamicin sulphate, for PMMA bone cement.

The suhB gene of Burkholderia cenocepacia is required for protein secretion, biofilm formation, motility and polymyxin B resistance

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of Gram-negative opportunistic pathogens that cause severe lung infections in patients with cystic fibrosis and display extreme intrinsic resistance to antibiotics including antimicrobial peptides. B. cenocepacia BCAL2157 encodes a protein homologous to SuhB, an inositol-1-monophosphatase from Escherichia coli, which was suggested to participate in posttranscriptional control of gene expression. In this work we show that a deletion of the suhB-like gene in B. cenocepacia (?suhBBc) was associated with pleiotropic phenotypes. The ?suhBBc mutant had a growth defect manifested by an almost 2-fold increase in the generation time relative to the parental strain. The mutant also had a general defect in protein secretion, motility and biofilm formation. Further analysis of the Type-2 and the Type-6 secretion systems activities revealed that these secretion systems were inactive in the ?suhBBc mutant. In addition, the mutant exhibited increased susceptibility to polymyxin B but not to aminoglycosides like gentamicin and kanamycin. Together, our results demonstrate that suhBBc deletion compromises general protein secretion including the activity of T2SS and T6SS, and affects polymyxin B resistance, motility, and biofilm formation. The pleiotropic effects observed upon suhBBc deletion demonstrate that suhBBc plays a critical role in the physiology of B. cenocepacia.

Intracellular survival of Burkholderia cepacia complex isolates in the presence of macrophage cell activation

Strains of the Burkholderia cepacia complex have emerged as a serious threat to patients with cystic fibrosis due to their ability to infect the lung and cause, in some patients, a necrotizing pneumonia that is often lethal. It has recently been shown that several strains of the B. cepacia complex can escape intracellular killing by free-living amoebae following phagocytosis. In this work, the ability of two B. cepacia complex strains to resist killing by macrophages was explored. Using fluorescence microscopy, electron microscopy and a modified version of the gentamicin-protection assay, we demonstrate that B. cepacia CEP021 (genomovar VI), and Burkholderia vietnamiensis (previously B. cepacia genomovar V) CEP040 can survive in PU5-1.8 murine macrophages for a period of at least 5 d without significant bacterial replication. Furthermore, bacterial entry into macrophages stimulated production of tumour necrosis factor and primed them to release toxic oxygen radicals following treatment with phorbol myristoyl acetate. These effects were probably caused by bacterial LPS, as they were blocked by polymyxin B. Infected macrophages primed with interferon gamma produced less nitric oxide than interferon-gamma-primed uninfected cells. We propose that the ability of B. cepacia to resist intracellular killing by phagocytic cells may play a role in the pathogenesis of cystic fibrosis lung infection. Our data are consistent with a model where repeated cycles of phagocytosis and cellular activation without bacterial killing may promote a deleterious inflammatory response causing tissue destruction and decay of lung function.