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Background Multidrug resistant Acinetobacter baumannii (MDR-Ab) is a Gram-negative bacterium known for causing severe nosocomial infections, attributed in part to its formation of biofilm. Siderophore is a virulence factor known to support biofilm formation by regulating iron availability. In this study, we screened 44 isolates of MDR-Ab from our Gram-negative repository to determine the strains that phenotypically form biofilm and produce siderophore. The results were compared to Pseudomonas aeruginosa PAO1, which produces both biofilm and siderophore. Methods Isolates were grown overnight in minimal M9 medium supplemented with casamino acids and hydroxyquinones at 37°C. Bacterial cells were normalized (to OD 600=0.01) and a standard diluted 10-3 tube was used in the study. A 96-well plate was inoculated with 100 microliters of each isolate in quadruplicates. This process was repeated in Tygon tubes with 50 microliters of each isolate in triplicates. The plate and Tygon tubes were incubated statically for 48 hours at 30°C and then stained with crystal violet. The contents were dissolved in 33% glacial acetic acid and analyzed by spectrophotometry to measure biofilm formation. Siderophore secretion was measured in supernatants with Chrome Azurol S (CAS) reagent and production was observed on CAS agar plates. Results High levels of biofilm formation were observed in 8 strains of MDR-Ab in the 96-well plate (3, 4, 9, 22, 61, 1010, 1012, 1022) and 6 strains in Tygon tubes (3, 4, 16, 66, 1002, 1010) (Fig. 1). There was minimal siderophore production in MDR-Ab isolates compared to PAO1 in both the 96-well plate and Tygon tubes (Fig. 2). Only 4 strains lacked siderophore production on CAS agar and were inversely negative for the secretion in medium. Figure 1 Biofilm formation in a 96-well plate and Tygon tubes (A) High levels of biofilm formation were observed in MDR-Ab strain numbers 3, 4, 9, 22, 61, 1010, 1012, 1022 in the 96-well plate. (B) High levels of biofilm formation were observed in MDR-Ab strain numbers 3, 4, 16, 66, 1002, 1010 in Tygon tubes. Figure 2 Degree of siderophore production in a 96-well plate and Tygon tubes Siderophore production of MDR-Ab was limited compared to PAO1 after inoculation in a 96-well plate (A) and in Tygon tubes (B). Conclusion Many strains of MDR-Ab readily form biofilm. Overall siderophore production is lower in MDR-Ab compared to consistent production by PAO1, but this does not appear to affect MDR-Ab’s ability to form biofilm. Unlike in PAO1, biofilm formation in MDR-Ab may occur independently of siderophore production. This research serves as a basis for understanding future MDR-Ab biofilm elimination in patient catheters and indwelling devices. Disclosures All Authors: No reported disclosures

Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

Brucella abortus has been shown to produce two siderophores: 2,3-dihydroxybenzoic acid (2,3-DHBA) and brucebactin. Previous studies on Brucella have shown that 2,3-DHBA is associated with erythritol utilization and virulence in pregnant ruminants. The biosynthetic pathway and role of brucebactin are not known and the only gene shown to be involved so far is entF. Using cre-lox methodology, an entF mutant was created in wild-type B. abortus 2308. Compared with the wild-type strain, the ΔentF strain showed significant growth inhibition in iron minimal media that became exacerbated in the presence of an iron chelator. For the first time, we have demonstrated the death of the ΔentF strain under iron-limiting conditions in the presence of erythritol. Addition of FeCl(3) restored the growth of the ΔentF strain, suggesting a significant role in iron acquisition. Further, complementation of the ΔentF strain using a plasmid containing an entF gene suggested the absence of any polar effects. In contrast, there was no significant difference in survival and growth between the ΔentF and wild-type strains grown in the murine macrophage cell line J774A.1, suggesting that an alternate iron acquisition pathway is present in Brucella when grown intracellulary.

Brucella spp. are Gram-negative, facultative intracellular bacterial pathogens that cause abortion in livestock and undulant fever in humans worldwide. Brucella abortus strain 2308 is a pathogenic strain that affects cattle and humans. Currently, there are no efficacious human vaccines available. However, B. abortus strain RB51, which is approved by the USDA, is a live-attenuated rough vaccine against bovine brucellosis. Live strain RB51 induces protection via CD4+ and CD8+ T-cell-mediated immunity. To generate an optimal T-cell response, strong innate immune responses by dendritic cells (DCs) are crucial. Because of safety concerns, the use of live vaccine strain RB51 in humans is limited. Therefore, in this study, we analyzed the differential ability of the same doses of live, heat-killed (HK) and γ-irradiated (IR) strain RB51 in inducing DC activation and function. Smooth strain 2308, live strain RB51 and lipopolysaccharide were used as controls. Studies using mouse bone marrow-derived DCs revealed that, irrespective of viability, strain RB51 induced greater DC activation than smooth strain 2308. Live strain RB51 induced significantly (P≤0.05) higher DC maturation than HK and IR strains, and only live strain RB51-infected DCs (at multiplicity of infection 1 : 100) induced significant (P≤0.05) tumor necrosis factor-α and interleukin-12 secretion.

Brucellosis caused by Brucella species is reportedly the most common zoonotic infection worldwide. The bacterial pathogen is also classified by the Centers for Disease Control and Prevention as a category (B) pathogen that has the potential for development as a bioweapon. Although eight genomes of Brucella have been sequenced, little information is available regarding the regulation of gene expression and promoter activity in Brucella spp. We therefore constructed a set of broad-host-range vectors expressing the lacZ reporter gene from various promoters. Four groups of promoters (Brucella native, antibiotic resistant, bacteriophage and synthetic promoters) were tested in vivo and in vitro in Brucella suis. The highest level of heterologous gene expression was achieved with synthetic hybrid trc promoter carrying the adenine-rich upstream element. Furthermore, this demonstrates the usefulness of synthetic promoters for enhanced level of gene expression in Brucella spp.

A 97-kDa purified aminopeptidase N (PepN) of Brucella melitensis was previously identified to be immunogenic in humans. The B. melitensis pepN gene was cloned, expressed in Escherichia coli and purified by affinity chromatography. The recombinant PepN (rPepN) exhibited the same biochemical properties, specificity and susceptibility to inhibitors as the native PepN. rPepN was evaluated as a diagnostic antigen in an indirect enzyme-linked immunosorbent assay (ELISA) using sera from patients with acute and chronic brucellosis. The specificity of the ELISA was determined with sera from healthy donors. The ELISA had a cutoff value of 0.156 with 100% specificity and 100% sensitivity. Higher sensitivity was obtained using rPepN compared with crude extract from B. melitensis. Anti-PepN sera did not exhibit serological cross-reaction to crude extracts from Rhizobium tropici, Ochrobactrum anthropi, Yersinia enterocolitica 09 or E. coli O157H7.