Reduction of Staphylococcus aureus and Pseudomonas aeruginosa colonisation on PVC through covalent surface attachment of fluorinated thiols

OBJECTIVES: This study reports the development, characterisation and microbiological testing of surface-modified polyvinylchloride (PVC) films for the purpose of reducing bacterial adherence.

METHODS: Irreversible covalent surface modification was achieved via nucleophilic substitution of fluorinated thiol-terminated compounds onto the polymer backbone. Four fluorinated modifiers, 2,3,5,6-tetrafluorothiophenol (TFTP), 4-(trifluoromethyl)thiophenol (TFMTP), 3,5-bis(trifluoromethyl)benzenethiol (BTFMBT) and 3,3,4,4,5,5,6,6,7, 7,8,8,9,9,10,10,10-heptadecafluoro-decane-1-thiol (HDFDT), were investigated. Modification was confirmed using attenuated total reflectance infrared spectroscopy; Raman mapping demonstrated that modification was homogenous on the macroscopic scale. The influence of fluorination on surface hydrophobicity was studied by contact angle analysis. The effect on microbial adherence was examined using Pseudomonas aeruginosa and Staphylococcus aureus.

KEY FINDINGS: The resultant changes in contact angle relative to control PVC ranged from -4 degrees to +14 degrees . In all cases, adherence of P. aeruginosa and S. aureus was significantly reduced relative to control PVC, with adherence levels ranging from 62% and 51% for TFTP-modified PVC to 32% and 7% for TFMTP-modified PVC.

CONCLUSIONS: These results demonstrate an important method in reducing the incidence of bacterial infection in PVC medical devices without compromising mechanical properties.

Inhibitor profiling of the Pseudomonas aeruginosa virulence factor LasB using N-alpha mercaptoamide template-based inhibitors

We report on the synthesis and biological evaluation of a focussed library of N-alpha mercaptoamide containing dipeptides as inhibitors of the zinc metallopeptidase Pseudomonas aeruginosa elastase (LasB, EC 3.4.24.26). The aim of the study was to derive an inhibitor profile for LasB with regard to mapping the S´1 binding site of the enzyme. Consequently, a focussed library of 160 members has been synthesised, using standard Fmoc-solid phase methods (on a Rink-amide resin), in which a subset of amino acids including examples of those with basic (Lys, Arg), aromatic (Phe, Trp), large aliphatic (Val, Leu) and acidic (Asp, Glu) side-chains populated the P´2 position of the inhibitor sequence and all 20 natural amino acids were incorporated, in turn, at the P´1 position. The study has revealed a preference for aromatic and/or large aliphatic amino acids at P´1 and a distinct bias against acidic residues at P´2. Ten inhibitor sequences were discovered that exhibited sub to low micromolar Ki values.

Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro

An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable. We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method. There was good agreement in interpretation of results between the two methods for both P. aeruginosa (90%) and B. cepacia complex isolates (70%) with tobramycin and for P. aeruginosa isolates (70%) with ceftazidime. As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method. The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed. (C) 2004 Elsevier B.V. All rights reserved.

Rapid colorimetric assay for antimicrobial susceptibility testing of Pseudomonas aeruginosa

A colorimetric assay based on the reduction of a tetrazolium salt {2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT)} for rapidly determining the susceptibility of Pseudomonas aeruginosa isolates to bactericidal antibiotics is described. There was excellent agreement between the tobramycin and ofloxacin MICs determined after 5 h using the XTT assay and after 18 h using conventional methods. The data suggests that an XTT-based assay could provide a useful method for rapidly determining the susceptibility of P. aeruginosa to bactericidal antibiotics.

Genomes of "phiKMV-like viruses" of Pseudomonas aeruginosa contain localized single-strand interruptions

The "phiKMV-like viruses" comprise an important genus of T7 related phages infecting Pseudomonas aeruginosa. The genomes of these bacteriophages have localized single-strand interruptions (nicks), a distinguishing genomic trait previously thought to be unique for T5 related coliphages. Analysis of this feature in the newly sequenced phage fkF77 shows all four nicks to be localized on the non-coding DNA strand. They are present with high frequencies within the phage population and are introduced into the phage DNA at late stages of the lytic cycle. The general consensus sequence in the nicks (5'-CGACxxxxxCCTAoh pCTCCGG-3') was shown to be common among all phiKMV-related phages.

Incidence and risk factors for pulmonary exacerbation treatment failures in cystic fibrosis patients chronically infected with Pseudomonas aeruginosa

Background: Pulmonary exacerbations (PEx) are responsible for much of the morbidity and mortality associated with cystic fibrosis (CF). However, there is a paucity of data on outcomes in CF PEx and factors influencing outcomes.

Methods: We reviewed all PEx in patients infected with Pseudomonas aeruginosa treated with parenteral antibiotics over 4 years at our center. Treatment failures were categorized a priori as those PEx requiring antibiotic regimen change, prolongation of therapy > 20 days because of failure to respond, an early recurrent event within < 45 days, or failure to recover lung function to > 90% of baseline FEV1.

Results: A total of 101 patients were followed for 452 PEx. Treatment failures were observed in 125 (28%) of PEx; antibiotic regimen change was observed in 27 (6%), prolongation of therapy in 29 (6%), early recurrent events in 63 (14%), and failure to recover lung function to > 90% of baseline FEV1 in 66 (15%). Demographic factors associated with one or more treatment failures per year included advanced airways disease, use of enteric feeds, CF-related diabetes, and CF liver disease but did not include female sex or F508del homozygosity. Increased treatment failure risk was associated with lower admission FEV1 and increased markers of inflammation. At therapeutic completion, increased inflammatory markers correlated with treatment failure. Failure rates decreased with increasing number of active antimicrobial agents used based on in vitro susceptibility (zero, 28/65 [43%]; one, 38/140 [27%]; two, 59/245 [24%]; three, 0/2 [0%]; P = .02).

Conclusions: One-fourth of PEx fail to respond adequately to initial management. Patient demographic and episode-specific clinical information can be used to identify individuals at increased risk of initial management failure.

Novel Inhibitors of the Pseudomonas aeruginosa Virulence Factor LasB: a Potential Therapeutic Approach for the Attenuation of Virulence Mechanisms in Pseudomonal Infection

Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial bio?lm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed “second-generation” antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB,N-mercaptoacetyl-Phe-Tyr-amide (Ki 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in bio?lm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal bio?lms, and to eradicate bio?lm completely when used in combination with conventional antibiotics.