Jacqueline Lagacé

Development of a Rapid and Sensitive Test for Identification of Major Pathogens in Bovine Mastitis by PCR

ABSTRACT Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli,Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae,Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 × 102 CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 × 103 CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.

Demonstration of a fusion mechanism between a fluid bactericidal liposomal formulation and bacterial cells.

It was previously demonstrated that fluid liposomal-encapsulated tobramycin, named Fluidosomes, was successful in eradicating mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection, whereas free antibiotic did not reduce colony-forming unit (CFU) counts (C. Beaulac et al., Antimicrob. Agents Chemother. 40 (1996) 665-669; C. Beaulac et al., J. Antimicrob. Chemother. 41 (1998) 35-41). These liposomes were also shown to be bactericidal in in vitro tests against strong resistant P. aeruginosa 64 microg/ml). The time needed to reach the maximal fusion rate was about 5 h for the resistant strain comparatively to much shorter time for the sensitive strain. The specific characteristics of Fluidosomes could help overcome bacterial resistance related to permeability barrier and even enzymatic hydrolysis considering the importance of synergy in the whole process of antibiotic resistance.

Aerosolization of Low Phase Transition Temperature Liposomal Tobramycin as a Dry Powder in an Animal Model of Chronic Pulmonary Infection Caused by Pseudomonas aeruginosa

Eradication of mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection has been previously demonstrated following the intratracheal administration of Fluidosomes, a low phase transition temperature (low T(C)) liposomal tobramycin preparation administered in liquid form (Beaulac et al., Antimicrob. Agents Chemother., 40, 665-669, 1996). In the present work, the same liposomal formulation was administered as a dry powder aerosol to an animal model of chronic pulmonary infection in view of a possible clinical development in cystic fibrosis patients. Chronic infection was established by intratracheal administration of 10(5) cfu of a mucoid variant of P. aeruginosa, PA 508, prepared in agar beads. Sixteen hours after one aerosol treatment, the cfu counts performed on lungs (pair) treated with liposomal tobramycin were of 4.31x10(5) cfu/lungs comparatively to 1.32x10(8) and 3.02x10(8) cfu/lungs respectively in untreated and in lungs treated with free antibiotic. Considering the quantity of liposome-tobramycin that has reached the lungs, the results suggest that aerosolization of low T(C) liposomal tobramycin used as a dry powder preparation could be an effective way of treating chronic pulmonary infection caused by Pseudomonas.

In Vitro Kinetics of Drug Release and Pulmonary Retention of Microencapsulated Antibiotic in Liposomal Formulations in Relation to the Lipid Composition

In previous in-vivo studies, we demonstrated that liposomal entrapment of tobramycin resulted in an increased availability of the antibiotic in the lungs without increasing bactericidal efficacy (Omri et al. 1994). With the aim of developing liposomal formulations allowing more efficient liposome-bacteria interactions, we studied the influence of lipid composition on both drug release and pulmonary retention of encapsulated tobramycin. The phase transition temperatures of nine liposome-tobramycin formulations consisting of two synthetic phospholipids (distearoyl phosphatidylcholine (DPSC) or dipalmitoyl phosphatidylcholine (DPPC) with dimyristoyl phosphatidyl-glycerol (DPMG) or dimyristoyl phosphatidylcholine (DMPC) were determined by differential scanning calorimetry. Liposomes, varying in terms of membrane fluidity and charge were submitted to in-vitro and in-vivo kinetic studies while retention and release of tobramycin were measured by high-performance liquid chromatography (HPLC). Five less fluid liposome formulations showed absence or very low tobramycin release in in-vitro tests and long term pulmonary retention of tobramycin. Four fluid liposome formulations showed in vitro tests modulated tobramycin release while pulmonary retention of tobramycin was dependent of the presence of charged phospholipids. Administration of charged fluid liposomes in mice showed a low level of tobramycin in the kidneys; non-charged fluid liposomes exhibited a relatively high level of tobramycin retention in the kidneys.

Liposome-encapsulated antibiotics: preparation, drug release and antimicrobial activity against Pseudomonas aeruginosa

Ticarcillin- and tobramycin-resistant strains of Pseudomonas aeruginosa were shown to have a markedly increased sensitivity to antibiotics enclosed in liposomes. This was demonstrated by growth inhibition of two resistant P. aeruginosa strains in the presence of the liposome-enclosed ticarcillin and tobramycin at 2 per cent and 20 per cent of their respective minimum inhibitory concentration. The liposome-enclosed antibiotic was as effective against the beta-lactamase-producing strain as against the non-beta-lactamase producing strain. Entrapment efficiency of the two antibiotics with the dehydration-rehydration vesicle (DRV) method was largely superior to other methodologies used. Kinetic studies with DRV demonstrated that tobramycin and ticarcillin-loaded liposomes still contained 83 per cent and 67 per cent of drug respectively after 24 h at 37 degrees C.

Evaluation of the pulmonary and systemic immunogenicity of Fluidosomes, a fluid liposomal–tobramycin formulation for the treatment of chronic infections in lungs

In previous studies, we have developed a fluid bactericidal liposomal formulation containing tobramycin, called Fluidosomes, which has been shown to be highly bactericidal both in in vitro and in in vivo studies against Pseudomonas aeruginosa and other related and unrelated bacteria. One foreseeable application of these Fluidosomes is the treatment of chronic pulmonary infections in cystic fibrosis patients colonized with P. aeruginosa and other related bacteria. Considering the capacity of some liposomal preparations to play an adjuvant role in vaccines, the non-immunogenicity of Fluidosomes has to be demonstrated. The systemic and local immunogenicity of Fluidosomes were assessed by effectuating repeated intraperitoneal (i.p.) and intratracheal (i.t. ) immunizations in BALB/c mouse. No significant mucosal and serum immune responses against Fluidosomes and/or tobramycin were detected as compared with preimmune sera. These data suggest that Fluidosomes could be administered repeatedly without adverse immune responses to control chronic pulmonary infections in cystic fibrosis.

Aminoglycoside detection using a universal ELISA binding procedure onto polystyrene microtiter plates in comparison with HPLC analysis and microbiological agar-diffusion assay

The use of enzyme-linked immunosorbent assay for the detection of aminoglycosides has been hindered due to low molecular weight compound adsorption to solid phases. Here, we describe an enzyme-linked immunosorbent assay based on the treatment of polystyrene microtiter plates with Alcian blue prepared in acetic acid prior to coating with the antibiotic. Whereas no detection of tobramycin was possible on commercially treated or untreated enzyme-linked immunosorbent assay plates, the Alcian blue treatment permitted detection of 0.025 and 0.05 microg ml(-1) of tobramycin respectively using 0.05 and 0.1% of Alcian blue with a coefficient of variation of 1.85 and 7.69%, respectively. Comparative studies of five tobramycin samples of unknown quantity using enzyme-linked immunosorbent assay and high-performance liquid chromatography gave equivalent results while those done via microbiological agar-diffusion assay were an overestimation of the actual quantity. The use of the Alcian blue pretreatment enzyme-linked immunosorbent assay procedure has permitted, in previous studies, the measure of antibodies against synthetic peptides and phospholipids. Subsequently, our demonstration of the sensitivity and reliability of this method in the quantification of tobramycin strongly suggests that the use of Alcian blue pretreatment in enzyme-linked immunosorbent assay can be applied universally to avert molecule immobilization problems on solid phases.

Measurement of mouse anti-phospholipid antibodies to solid-phase microspheres by both flow cytofluorometry and Alcian blue-pretreated microtitre plates in an ELISA

Conventional solid-phase immunoassays measuring interactions between anti-phospholipid antibodies and phospholipids are generally characterized by problems of reproducibility and high levels of non-specific binding. Here we describe two immunoassays based on the use of phospholipids in the form of solid-phase microspheres to measure the presence of anti-phospholipid antibodies in sera. Following the production of antibodies in mice against liposomes containing lipid A, we show that flow cytofluorometric analysis provides a reproducible and sensitive way to detect anti-phospholipid antibodies. We also present a sensitive, rapid and reproducible enzyme-linked immunosorbent assay (ELISA) using Alcian blue pretreated microtitre plates and solid-phase microspheres as coating antigen. This ELISA permitted the detection of antibodies to 1/1000 dilution, while untreated plates gave negative results. Such modified ELISA procedures may be applicable to other types of molecule exhibiting solid-phase binding problems e.g. synthetic peptides (J. Immunol. Methods 175 (1994) 131-135).

Alcian blue-treated polystyrene microtitre plates for use in an ELISA to measure antibodies against synthetic peptides.

Short peptides often do not bind well to the plastic surface of microtitre wells in solid-phase immunoassays. To improve the reactivity of synthetic peptides coated on the solid phase, we have developed a highly sensitive, rapid and simple ELISA. This ELISA is based on the treatment of microtitre plates with Alcian blue prepared in acetic acid prior to coating the target peptide. With hyperimmune serum, this treatment increases the specific signal in such a manner that the detection of antibodies needs less antigen than with conventional ELISA. Moreover, the Alcian blue treatment was shown to be particularly effective for the detection of monoclonal antibodies against a synthetic peptide derived from the human immunodeficiency type 1 (HIV-1) vpu protein. These monoclonal antibodies that were not detected in conventional ELISA gave a positive signal up to a 1 in 1000 dilution after Alcian blue treatment of microtitre wells. This assay should be applicable to a variety of synthetic peptides and other types of molecules posing problems in assays requiring their immobilization on solid phases.

In Vitro Bactericidal Evaluation of a Low Phase Transition Temperature Liposomal Tobramycin Formulation as a Dry Powder Preparation Against Gram Negative and Gram Positive Bacteria

AbstractIn previous studies, delivery of a liquid preparation of encapsulated tobramycin in fluid liposomes, called Fluidosomes, has showed a marked improvement in the bactericidal activity against in-vitro and in-vivo extracellular infections. To examine the possibility of developing aerosol treatment using dehydrated Fluidosomes for the treatment of chronic pulmonary infections, freeze-dried preparations of tobramycin and Fluidosomes were tested against cultures of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burk-holderia cepacia, Escherichia coli and Staphylococcus aureus. Bacterial colonies were enumerated 0, 1, 3, 6 and 16 h after the addition of the antibiotic. Sixteen hours post-treatment, the growth of P. aeruginosa, S. maltophilia, B. cepacia and E. coli in the presence of sub-minimal inhibitory concentrations of tobramycin was significantly lowered respectively by 17 (P < 0.01), 40 (P < 0.001), 47 (P < 0.001), and 50 (P < 0.001) times in comparison with growth in the presence of free a...