Antibacterial activity of Lactobacillus acidophilus and Lactobacillus casei against methicillin-resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant microorganism and the principal nosocomial pathogen worldwide. The antibacterial activity of lactic acid bacteria against MRSA from ten human clinical isolates as well as MRSA standard strain ATCC 43300 was tested in vitro. The Lactobacillus (Lb.) strains (Lb. acidophilus CL1285(®) and Lb. casei LBC80R) as pure cultures, which came from commercial food products were employed. The growth inhibitory effect produced by the antimicrobial activity of the lactic acid bacteria on the MRSA strains was tested on solid medium using agar diffusion methods as well as a using a liquid medium procedure that contained a mixture of MRSA and lactic acid bacteria cultures. In the latter instance, we were able to demonstrate that the direct interaction of lactic acid bacteria and MRSA in such a mixture led to the elimination of 99% of the MRSA cells after 24 h of their incubation at 37°C.

The Effect of Various Probiotic Strains or Avilamycin Feed Additive on Immune Defense Markers and Acute-Phase Response to Salmonella Infection in Chickens.

Probiotics are a nutritional tool for disease prevention. It has been proposed that stimulation of immune response could affect the growth-promoting properties of antimicrobial growth promoters as well as the control of foodborne pathogens. The current study compares immune response in the blood of 280 non-infected and Salmonella-infected chickens fed either with the growth promoter avilamycin or with one of five probiotic strains of Lactobacillus and Bifidobacterium, which also showed growth-promoting properties. All of the probiotic strains stimulated superoxide anion production and the proliferation of leukocytes, while raising lysozyme and γ-globulin levels (by up to 65%, p < 0.01), which are important factors in native and cell-mediated immune defense against pathogens. In contrast, among the two strains examined, specific Salmonella antibodies were induced only by L. salivarius, and not by B. animalis, as assessed by the ELISA method and confirmed by an agglutination reaction (p < 0.05). In the avilamycin-fed group, both non-infected and infected chickens showed decreased levels of these immune markers (by 30%) and increased levels of ceruloplasmin by up to 35%. In contrast, the probiotics suppressed acute-phase response assessed by ceruloplasmin by up to 32%. This correlation implies that various antimicrobial feed additives have a distinct effect on immunomodulation, which may affect different mechanisms in the nutrition-related metabolism associated with the rate of weight gain in chickens. The data could contribute to the design of innovative antimicrobial feed additives in the food industry and consequently to well-being of humans.

In vitro expression of the restriction endonucleases LlaMI and ScrFI isolated from Lactococcus lactis M19 and UC503.

A new restriction endonuclease LlaMI has been characterized in Lactococcus lactis subsp. cremoris M19. LlaMI recognizes the sequence 5'-CCNGG-3' and cuts after the second cytosine. This restriction endonuclease is related to commercially available ScrFI but not identical to it. Comparative analysis of the predicted amino acid sequences of LlaMI and ScrFI indicates five non-conservative amino acid changes between these two restriction enzymes. These two enzymes were expressed in vitro as histidine-tagged fusion proteins. LlaMI was shown to be more sensitive to high salt concentration than ScrFI. Southern blotting and hybridization analysis indicate that the gene for LlaMI R/M system is chromosomally encoded.

A rapid method combining immunofluorescence and flow cytometry for improved understanding of competitive interactions between lactic acid bacteria (LAB) and methicillin-resistant S. aureus (MRSA) in mixed culture.

The increasing frequency of methicillin-resistant Staphylococcus aureus (MRSA) infections in hospital and community settings highlights the need for effective anti-MRSA agents that will not contribute to the growing problem of antibiotic resistance. Lactic acid bacteria (LAB) are known to exclude various pathogens through multiple mechanisms. In vitro models studying interactions of pathogens and LAB in mixed cultures use selective agar plates to quantify changes in target populations. We applied commercially available S. aureus-specific polyclonal antibodies conjugated with fluorescein isothiocyanate (FITC) for this purpose, producing a bright green signal that clearly differentiates S. aureus from LAB species when mixed cultures are analyzed by flow cytometry and fluorescent microscopy. Flow cytometry of mixed cultures revealed a much larger population of MRSA cells than was detectable using selective agar plates. To our knowledge, this is the first time immunofluorescent flow cytometry has been applied to the study of competitive exclusion in mixed bacterial populations over time.

Purification and separation of hydrophobic serum amyloid A precursor isoforms by a one-step preparative method.

The levels of two major serum amyloid A precursor isoforms, SAA1 and SAA2, which are associated with high-density lipoproteins (HDL) are increased during inflammation. The hydrophobic character and the small size difference--corresponding to just 0.8 kDa--between these two members of the SAA family hinder their separation and purification on a large scale by conventional methods. In the current work, both mouse SAA proteins were purified from HDL-SAA and acute-phase serum within 10 h in a one-step procedure using the high-resolution, continuous-elution preparative gel electrophoresis Prep-Cell system in combination with Tris/Glycine SDS-PAGE. Moreover, applying the Tris/Tricine system on the Prep-Cell resulted not only in purification of the SAA proteins, but also in their separation within 16 h. The SAA isoforms were freed from SDS using a Centricon concentrator and were identified using monoclonal antibodies. Optical density profile plots of gel protein or Western blot bands in combination with a colorimetric spectrophotometric protein assay showed that the recovery of the isoforms ranged from 38% to 60%. These results show that the preparative gel electrophoresis system Prep-Cell is a suitable device for separating SAA1 and SAA2 proteins in a simplified, convenient, and fast procedure, which can be applied on a small or large scale.