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 ؋ 10 2 CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 ؋ 10 3 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.Bovine mastitis (BM) is an inflammation of the mammary gland, usually due to a microbial infection (28), which causes North American dairy producers to lose billions of dollars every year. These losses are primarily due to lower milk yields, reduced milk quality, and higher production costs. BM often becomes chronic, and it is important to identify quickly the new clinical cases in order to control infection in the herd. The bacteria responsible for BM can be classified as environmental (Escherichia coli, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis) or contagious (Staphylococcus aureus and Streptococcus agalactiae) depending of their primary reservoir (environment versus infected mammary gland quarter) (11,25).The suitability of a detection method for routine diagnosis depends on several factors, such as specificity, sensitivity, expense, amount of time, and applicability to large numbers of milk samples. The most common but unspecific method (2) to identify potential chronic infections is a somatic cell count: the California Mastitis Test in field conditions and the automated method in the diagnosis laboratory. Currently, the method of identification of the mammary gland pathogens is by in vitro culture, which provides the "gold standard"; however, this technique is labor-intensive and time-consuming. Two other problems can be encountered when these methods of identification are used: first, 2 to 3 days are required to grow, isolate, and identify the pathoge...
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.
Antisense therapy for the treatment of bacterial infections is a very attractive alternative to overcome drug resistance problems. However, the penetration of antisense oligonucleotides into bacterial cells is a major huddle that has delayed research and application in this field. In the first part of this study, we defined efficient conditions to encapsulate plasmid DNA and antisense oligonucleotides in a fluid negatively charged liposome. Subsequently, we evaluated the potential of liposome-encapsulated antisense oligonucleotides to penetrate the bacterial outer membrane and to inhibit gene expression in bacteria. It was found that 48.9+/-12% and 43.5+/-4% of the purified plasmid DNA and antisense oligonucleotides were respectively encapsulated in the liposomes. Using fluorescence-activated cell sorting analysis, it was shown, after subtraction of the fluorescence values due to the aggregation phenomenon measured at 4 degrees C, that about 57% of bacterial cells had integrated the encapsulated antisense oligonucleotides whereas values for free antisenses were negligible. The uptake of the encapsulated anti-lacZ antisense oligonucleotides resulted in a 42% reduction of beta-galactosidase compared to 9% and 6% for the encapsulated mismatch antisense oligonucleotides and the free antisense oligonucleotides respectively. This work shows that it is possible to encapsulate relatively large quantities of negatively charged molecules in negative fluid liposomes and suggests that fluid liposomes could be used to deliver nucleic acids in bacteria to inhibit essential bacterial genes.
It has been shown previously that tobramycin encapsulated in fluid liposomes (composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG)) eradicated mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection. Exponential cultures of P. aeruginosa, Stenotrophomonas maltophila, Burkholderia cepacia, Escherichia coli and Staphylococcus aureus were treated with (i) free tobramycin, (ii) sub-MIC tobramycin encapsulated in DPPC/DMPG liposomes, (iii) control liposomes without antibiotic or (iv) control liposomes combined with free tobramycin. Bacterial colonies were counted 0, 1, 3, 6 and 16 h after addition of antibiotic. After 3 h, the growth of B. cepacia, E. coli and S. aureus was reduced 129, 84 and 566 times respectively in cultures treated with encapsulated antibiotic compared with those treated with free antibiotic. Six hours and 16 h after treatment, the maximal reduction of growth between strains treated with liposome-encapsulated tobramycin and free tobramycin was 84, 129, 166, 10(5) and 10(4) times respectively for P. aeruginosa, B. cepacia, E. coli, S. maltophilia and S. aureus. The liposomes were stable at 4 degrees C and at room temperature for the whole period studied. At 37 degrees C, equivalent stability was observed for the first 16 h of the study. Administration of antibiotic encapsulated in DPPC/DMPG liposomes may thus greatly improve the management of resistant infections caused by a large range of microorganisms. The strong bactericidal activity of the encapsulated antibiotic at sub-MIC doses of the strains tested cannot be explained only as a result of prolonged residence time of liposome-encapsulated tobramycin and the resulting release of entrapped antibiotic at the bacterial site; rather, direct interaction of chemoliposomes and bacteria, probably by a fusion process, may explain the bactericidal effect of the sub-MIC antibiotic doses used.
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