BackgroundBiosurfactants (BS) are amphiphilic compounds produced by microbes, either on the cell surface or secreted extracellularly. BS exhibit strong antimicrobial and anti-adhesive properties, making them good candidates for applications used to combat infections. In this study, our goal was to assess the in vitro antimicrobial, anti-adhesive and anti-biofilm abilities of BS produced by Lactobacillus jensenii and Lactobacillus rhamnosus against clinical Multidrug Resistant (MDR) strains of Acinetobacter baumannii, Escherichia coli, and Staphylococcus aureus (MRSA). Cell-bound BS from both L. jensenii and L. rhamnosus were extracted and isolated. The surface activities of crude BS samples were evaluated using an oil spreading assay. The antimicrobial, anti-adhesive and anti-biofilm activities of both BS against the above mentioned MDR pathogens were determined.ResultsSurface activities for both BS ranged from 6.25 to 25 mg/ml with clear zones observed between 7 and 11 cm. BS of both L. jensenii and L. rhamnosus showed antimicrobial activities against A. baumannii, E. coli and S. aureus at 25-50 mg/ml. Anti-adhesive and anti-biofilm activities were also observed for the aforementioned pathogens between 25 and 50 mg/ml. Finally, analysis by electron microscope indicated that the BS caused membrane damage for A. baumannii and pronounced cell wall damage in S. aureus.ConclusionOur results indicate that BS isolated from two Lactobacilli strains has antibacterial properties against MDR strains of A. baumannii, E. coli and MRSA. Both BS also displayed anti-adhesive and anti-biofilm abilities against A. baumannii, E. coli and S. aureus. Together, these capabilities may open up possibilities for BS as an alternative therapeutic approach for the prevention and/or treatment of hospital-acquired infections.

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Entry of opsonized pathogens into phagocytes may benefit or, paradoxically, harm the host. Opsonization may trigger antimicrobial mechanisms such as reactive oxygen or nitric oxide (NO) production but may also provide a safe haven for intracellular replication. Brucellae are natural intramacrophage pathogens of rodents, ruminants, dogs, marine mammals, and humans. We evaluated the role of opsonins in Brucellamacrophage interactions by challenging cultured murine peritoneal macrophages with Brucella melitensis 16M treated with complement-and/or antibody-rich serum. Mouse serum rich in antibody against Brucella lipopolysaccharide (LPS) (aLPS) and human complement-rich serum (HCS) each enhanced the macrophage uptake of brucellae. Combinations of suboptimal levels of aLPS (0.01%) and HCS (2%) synergistically enhanced uptake. The intracellular fate of ingested bacteria was evaluated with an optimal concentration of gentamicin (2 g/ml) to control extracellular growth but not kill intracellular bacteria. Bacteria opsonized with aLPS and/or HCS grew equally well inside macrophages in the absence of gamma interferon (IFN-␥). Macrophage activation with IFN-␥ inhibited replication of both opsonized and nonopsonized brucellae but was less effective in inhibiting replication of nonopsonized bacteria. IFN-␥ treatment of macrophages with opsonized or nonopsonized bacteria enhanced NO production, which was blocked by N G -monomethyl L-arginine (MMLA), an NO synthesis inhibitor. MMLA also partially blocked IFN-␥-mediated bacterial growth inhibition. These studies suggest that primary murine macrophages have limited ability to control infection with B. melitensis, even when activated by IFN-␥ in the presence of highly opsonic concentrations of antibody and complement. Additional cellular immune responses, e.g., those mediated by cytotoxic T cells, may play more important roles in the control of murine brucellosis.Brucella spp., short, nonmotile, nonsporulating, nonencapsulated, gram-negative aerobic rods, are important facultative intracellular pathogens of humans and livestock. Brucella melitensis usually infects sheep, goats, and camels and is the most pathogenic species for humans (1). Like other facultative intracellular pathogenic bacteria (e.g., Francisella tularensis, Listeria monocytogenes, Mycobacterium spp., and Legionella pneumophila), clearance of Brucella infection relies on both cellmediated immunity (1,3,7,12,20,21,27,30,38) and humoral responses (10,22,29,35). The interplay of these two arms of the immune response, however, is not well understood.Successful infection of the host by Brucella reflects the ability of the bacterium to establish itself in an intracellular environment favorable for its replication. The presence of complement or antibody in the extracellular fluid favors killing of some Brucella strains (35,38). Opsonization by these humoral factors also enhances uptake by phagocytic cells that shelter the bacteria. The intracellular fate of brucellae may depend on the bacterial species or the kind of ph...

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of conjunctiva or traumatized skin by infected animal products. In addition, Brucella is recognized as a biowarfare threat agent. Although a vaccine to protect humans from natural or deliberate infection could be useful, vaccines presently used in animals are unsuitable for human use. We tested orally administered live, attenuated, purine auxotrophic B. melitensis WR201 bacteria for their ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M bacteria. Immunized mice made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 and gamma interferon when grown in cultures with Brucella antigens. Immunization led to protection from disseminated infection and enhanced clearance of the challenge inoculum from the lungs. Optimal protection required administration of live bacteria, was related to immunizing dose, and was enhanced by booster immunization. These results establish the usefulness of oral vaccination against respiratory challenge with virulent Brucella and suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis.Human brucellosis, caused mostly by Brucella abortus, B. melitensis, and B. suis, can be acquired by ingestion, inhalation, or contamination of conjunctiva or traumatized skin by infected animal products (3). Bacteria spread, presumably via lymphatics and blood (8), from the site of entry to the mononuclear phagocyte system. Although generalized symptoms of fever, sweating, and fatigue are nearly universal for patients with acute brucellosis, onset can be insidious and many patients present with or develop localized foci of infection, especially in the bones and joints (24). Control of brucellosis in domestic food animals has markedly reduced the incidence of human brucellosis in the United States, but the disease represents an important cause of morbidity worldwide. A human vaccine would be valuable for individuals who may be occupationally exposed to brucellae and for persons who consume unpasteurized dairy products from areas in which brucellae are endemic. In addition, Brucella species are recognized as biowarfare or bioterror threat agents by the Center for Disease Control, further supporting the need to develop effective medical protective measures against them.We have previously reported that levels of B. melitensis WR201, a purEK deletion mutant of B. melitensis 16M, are attenuated for growth in mononuclear phagocytes (5) and in mice (4) after intraperitoneal (i.p.) inoculation relative to parent strain results. Mice inoculated i.p. with strain WR201 make antibody directed against lipopolysaccharide (LPS) and Brucella protein, and their splenocytes produce gamma interferon (IFN-␥) and interleukin-2 (IL-2) when grown in cultures with Brucella antigens (11). In addition, immunization of mice by i.p. inoculation...

After intranasal inoculation, Brucella melitensis chronically infects the mononuclear phagocyte system in BALB/c mice, but it causes no apparent illness. Adaptive immunity, which can be transferred by either T cells or antibody from immune to naive animals, confers resistance to challenge infection. The role of innate, non-B-, non-T-cell-mediated immunity in control of murine brucellosis, however, is unknown. In the present study, we documented that BALB/c and C57BL/6 mice had a similar course of infection after intranasal administration of 16M, validating the usefulness of the model in the latter mouse strain. We then compared the course of infection in Rag1 knockout mice (C57BL/6 background) (referred to here as RAG-1 mice) which have no B or T cells as a consequence of deletion of Rag1 (recombination-activating gene 1), with infection in normal C57BL/6 animals after intranasal administration of B. melitensis 16M. C57BL/6 mice cleared brucellae from their lungs by 8 to 12 weeks and controlled infection in the liver and spleen at a low level. In contrast, RAG-1 mice failed to reduce the number of bacteria in any of these organs. From 1 to 4 weeks after inoculation, the number of splenic bacteria increased from 2 to 4.5 logs and remained at that level. In contrast to the consistently high numbers of brucellae observed in the spleens, the number of bacteria rose in the livers sampled for up to 20 weeks. Immunohistologic examination at 8 weeks after infection disclosed foci of persistent pneumonia and large amounts of Brucella antigen in macrophages in lung, liver, and spleen in RAG-1, but not C57BL/6, mice. These studies indicate that T-and B-cell-independent immunity can control Brucella infection at a high level in the murine spleen, but not in the liver. Immunity mediated by T and/or B cells is required for clearance of bacteria from spleen and lung and for control of bacterial replication in the liver.Brucellosis, a zoonosis that affects several species of domestic animals, manifests itself in humans as a systemic, febrile illness. Most human disease is caused by Brucella melitensis, but B. abortus and B. suis are also highly pathogenic. The disease is recognized in more than 100 countries, with an estimated one million new cases per year. Most cases occur as a result of occupational exposure to animals or ingestion of nonpasteurized dairy products (6). Laboratory workers exposed to the agent are also at high risk of infection. Brucellosis can be acquired through ingestion and through breaks in the skin; aerosol transmission also occurs (6). There are no suitably attenuated, well-characterized human vaccines available.To simulate infection by a mucosal or aerosol route of infection, we have recently established a murine model of brucellosis in which BALB/c mice are inoculated intranasally with B. melitensis 16M (14, 17). In this model, the organism infects the lung and disseminates to the blood, liver, and spleen. Both antibody and cellular immune effectors mediate control of dissemination and replication of ...