Intracellular Brucella Abortus Research Articles

Use of the quantum dot-labeled solid lipid nanoparticles for delivery of streptomycin and hydroxychloroquine: A new therapeutic approach for treatment of intracellular Brucella abortus infection

Brucellosis is considered one of the most important infectious diseases affecting any tissue and organ in the human body. Due to the intracellular pathogenesis of Brucella species, the use of conventional antibiotics for managing chronic brucellosis has several limitations. Therefore, the study focused on the use of solid lipid nanoparticles (SLN) to deliver streptomycin (STR) for intracellular infection, with or without the combination of hydroxychloroquine (HCQ) to evaluate if there might be a boost in the antibiotic effect when using the STR or STR-NPs alone. We used the double emulsion technique to synthesize Nano drug carriers; afterward, the physicochemical characteristics of synthesized Nano drug carriers were determined. The in vitro antibacterial activity of free drugs and Nano drug carriers were evaluated using well diffusion, broth microdilution assays (BMD), and murine macrophage-like cells cell line J774A.1. Additionally, acute and chronic phases of brucellosis were inducted into Wistar rats, and healing capacity of Nano drug carriers on liver and spleen tissues was compared with free drugs. The zeta potential of nanoparticles, means of size, Polydispersity Index (PDI), drugs loading, and encapsulation efficiency were 15.2 mV, 312.5 ± 26 nm, 0.433 ± 0.09, 16.6% and 89.5%, respectively. Well diffusion and BMD methods did not show a significantly differ between free drugs and nano drug carriers. However, the Nano drug carriers remarkably decreased the number of bacteria in the cell line compared to the free drugs. STR/HCQ-SLN enhanced the healing processes of the liver and spleen after brucellosis induction. STR/HCQ-SLN showed better inhibitory effects against the chronic phase of B. abortus infection in comparison to the STR-SLN, but this difference was not statistically significant. Using nanoplatforms to enhance conventional anti-brucellosis agents is promising, green and safe. Due to the continuous release of drugs, drugs increase their accumulation at the site of infection, causing a more significant effect on the chronic and acute phases of brucellosis.

Enhanced Antibacterial Activity of Doxycycline and Rifampicin Combination Loaded in Nanoparticles against Intracellular Brucella abortus

Brucellosis is a zoonotic disease that is prevalent in livestock animals. The bacteria reside inside the macrophage cells of the host. The WHO has endorseda combination treatment therapy for brucellosis against the conventional monotherapy to avoid relapse and resistance. Therefore, we developed nanoparticles incorporating doxycycline and rifampicin in combination. The aim of the study is to develop polymeric nanoparticles incorporating doxycycline as well as rifampicin and investigate the antibacterial activity of nanoparticles in U937 human macrophage cells infected with B. abortus. Polymeric nanoparticles were developed by the emulsion-solvent diffusion method, and characterization was performed. The nanoparticles with high entrapment efficiency of both the drugs were developed successfully. Scanning electron microscopy revealed a spherical morphology with a size ranging ~450nm, which can be easily engulfed by the macrophages. Zeta potential confirmed the colloidal stability. Differential scanning calorimetry and X-ray diffraction suggested amorphization of doxycycline and rifampicin in nanoparticles. Fourier transfer infrared spectroscopy could not confirm the interaction of drugs with AOT. In vitro haemolysis study confirmed the safety of nanoparticles (<10%) for IV administration. Further, nanoparticles revealed the sustained release of both drugs, which followed diffusion kinetics. Nanoparticles were found stable for 6 months as per WHO guidelines. The internalization study revealed nanoparticles could be easily uptaken by U-937 human macrophage cells. The efficacy study demonstrated significantly high antibacterial activity of nanoparticles as compared to free drug solution in U937 human macrophages cells infected with Brucella abortus. It can be concluded that the developed nanoparticles entrapping doxycycline and rifampicin combination can be considered as a promising delivery system for enhancing the antibacterial activity against Brucella abortus.

Substantial Protective Immunity Conferred by a Combination of Brucella abortus Recombinant Proteins against Brucella abortus 544 Infection in BALB/c Mice.

Chronic infection with intracellular Brucella abortus (B. abortus) in livestock remains as a major problem worldwide. Thus, the search for an ideal vaccine is still ongoing. In this study, we evaluated the protective efficacy of a combination of B. abortus recombinant proteins; superoxide dismutase (rSodC), riboflavin synthase subunit beta (rRibH), nucleoside diphosphate kinase (rNdk), 50S ribosomal protein (rL7/L12) and malate dehydrogenase (rMDH), cloned and expressed into a pMal vector system and DH5α, respectively, and further purified and applied intraperitoneally into BALB/c mice. After first immunization and two boosters, mice were infected intraperitoneally (IP) with 5 × 104 CFU of virulent B. abortus 544. Spleens were harvested and bacterial loads were evaluated at two weeks post-infection. Results revealed that this combination showed significant reduction in bacterial colonization in the spleen with a log protection unit of 1.31, which is comparable to the average protection conferred by the widely used live attenuated vaccine RB51. Cytokine analysis exhibited enhancement of cell-mediated immune response as IFN-γ is significantly elevated while IL-10, which is considered beneficial to the pathogen's survival, was reduced compared to control group. Furthermore, both titers of IgG1 and IgG2a were significantly elevated at three and four-week time points from first immunization. In summary, our in vivo data revealed that vaccination with a combination of five different proteins conferred a heightened host response to Brucella infection through cell-mediated immunity which is desirable in the control of intracellular pathogens. Thus, this combination might be considered for further improvement as a potential candidate vaccine against Brucella infection.

A Microarray-Based Analysis of Differentially Expressed Genes in Intracellular Brucella abortus 544 within Mdbk Cells

Background: Modulation of gene expression is a fundamental requirement for adaptation of intracellular Brucella abortus. Since most of the current understanding mainly focus on professional phagocytes and renal involvement is uncommon in brucellosis, our aim was to identify and analyze changes in B. abortus gene expression in response to intracellular environment within a bovine kidney cell line. Methodology: B. abortus RNA were isolated from Madin-Darby bovine kidney (MDBK) epithelial cells during replicative phase and the transcriptional profile of intracellular B. abortus was characterized using microarray analysis. Results and interpretation: The microarray analysis revealed a total of 1,623 differentially expressed genes of ≥ 2-fold–788 (25.44%, 788/3098) upregulated and 835 (26.95%,835/3098) down-regulated genes as compared with free-living Brucella. Among these identified genes, 81 and 185 were upregulated and down-regulated at ≥ 7-fold, respectively, showing a marked induction of genes involved in transcription and distinct repression of genes involved in translation, ribosomal structure and biosynthesis. Conclusion: The identified genes in this study may provide new insights into the molecular interactions between B. abortus and non-phagocytic bovine cell line, MDBK. In addition, several differentially highly expressed transcripts were hypothetical genes with unknown function and/or unclassified which require further characterization due to their potential contribution in the virulence and strategy of Brucella to survive and proliferate within the host.

Simultaneous RNA-seq based transcriptional profiling of intracellular Brucella abortus and B. abortus-infected murine macrophages

Brucella is a zoonotic pathogen that survives within macrophages; however the replicative mechanisms involved are not fully understood. We describe the isolation of sufficient Brucella abortus RNA from primary host cell environment using modified reported methods for RNA-seq analysis, and simultaneously characterize the transcriptional profiles of intracellular B. abortus and bone marrow-derived macrophages (BMM) from BALB/c mice at 24 h (replicative phase) post-infection. Our results revealed that 25.12% (801/3190) and 16.16% (515/3190) of the total B. abortus genes were up-regulated and down-regulated at >2-fold, respectively as compared to the free-living B. abortus. Among >5-fold differentially expressed genes, the up-regulated genes are mostly involved in DNA, RNA manipulations as well as protein biosynthesis and secretion while the down-regulated genes are mainly involved in energy production and metabolism. On the other hand, the host responses during B. abortus infection revealed that 14.01% (6071/43,346) of BMM genes were reproducibly transcribed at >5-fold during infection. Transcription of cytokines, chemokines and transcriptional factors, such as tumor necrosis factor (Tnf), interleukin-1α (Il1α), interleukin-1β (Il1β), interleukin-6 (Il6), interleukin-12 (Il12), chemokine C-X-C motif (CXCL) family, nuclear factor kappa B (Nf-κb), signal transducer and activator of transcription 1 (Stat1), that may contribute to host defense were markedly induced while transcription of various genes involved in cell proliferation and metabolism were suppressed upon B. abortus infection. In conclusion, these data suggest that Brucella modulates gene expression in hostile intracellular environment while simultaneously alters the host pathways that may lead to the pathogen's intracellular survival and infection.

Microarray-Based Identification of Differentially Expressed Genes in Intracellular Brucella abortus within RAW264.7 Cells

Brucella spp. is a species of facultative intracellular Gram-negative bacteria that induces abortion and causes sterility in domesticated mammals and chronic undulant fever in humans. Important determinants of Brucella’s virulence and potential for chronic infection include the ability to circumvent the host cell’s internal surveillance system and the capability to proliferate within dedicated and non-dedicated phagocytes. Hence, identifying genes necessary for intracellular survival may hold the key to understanding Brucella infection. In the present study, microarray analysis reveals that 7.82% (244/3334) of all Brucella abortus genes were up-regulated and 5.4% (180/3334) were down-regulated in RAW264.7 cells, compared to free-living cells in TSB. qRT-PCR verification further confirmed a >5-fold up-regulation for fourteen genes. Functional analysis classified araC, ddp, and eryD as to partake in information storage and processing, alp, flgF and virB9 to be involved in cellular processes, hpcd and aldh to play a role in metabolism, mfs and nikC to be involved in both cellular processes and metabolism, and four hypothetical genes (bruAb1_1814, bruAb1_0475, bruAb1_1926, and bruAb1_0292) had unknown functions. Furthermore, we constructed a B. abortus 2308 mutant Δddp where the ddp gene is deleted in order to evaluate the role of ddp in intracellular survival. Infection assay indicated significantly higher adherence and invasion abilities of the Δddp mutant, however it does not survive well in RAW264.7 cells. Brucella may survive in hostile intracellular environment by modulating gene expression.

Gentamicin-loaded microspheres for reducing the intracellular Brucella abortus load in infected monocytes.

The intracellular antibiotic efficiency of gentamicin-loaded microspheres in the context of Brucella-infected murine monocytes was examined in vitro with a view to developing improved therapies for the treatment of brucellosis. Biodegradable microspheres made of end-group capped and uncapped poly(lactide-co-glycolide) 50:50 (PLGA 50:50 and PLGA 50:50H) and containing gentamicin sulphate were used to target Brucella abortus-infected J774 monocyte-macrophages. The infected cells were treated with 15 micro g of free or microencapsulated gentamicin and the efficacy of the treatments was measured after 24 h. The particle sizes were below 8 micro m and in vitro release of gentamicin from the microspheres followed a continuous (PLGA 50:50H) or a multiphasic (PLGA 50:50) pattern over 50 days. Treatment with gentamicin microencapsulated into the end-group uncapped PLGA 50:50H microspheres, decreased significantly the number of intracellular bacteria (typically by 2 log(10)) in comparison with untreated infected cells. Addition of 2% poloxamer 188 to the microsphere dispersion medium further reduced the infection (3.5 log(10)). Opsonization of the particles with non-immune mouse serum had no effect on the antibacterial efficacy of the microspheres. End-group capped PLGA 50:50 type microspheres containing the antibiotic were less effective at reducing intracellular bacteria ( approximately 1 log(10) reduction), although addition of poloxamer 188 to the dispersion medium again enhanced their intracellular antibacterial activity. Placebo PLGA 50:50 and PLGA 50:50H microspheres had no bactericidal activity. The results indicate that PLGA 50:50-microencapsulated gentamicin sulphate may be suitable for efficient drug targeting and delivery to reduce intracellular Brucella infections.

Synergistic action of streptomycin with other antibiotics of intracellular Brucella abortus in vitro.

Richardson, Marvis (Michigan State University, East Lansing) and Jane N. Holt. Synergistic action of streptomycin with other antibiotics on intracellular Brucella abortus in vitro. J. Bacteriol. 84:638-646. 1962.-It has been found that streptomycin acts synergistically with penicillin and tetracycline to inhibit the growth of Brucella abortus within bovine cells cultured in vitro. Extracellular penicillin at 10 to 50 mug/ml was essentially bacteriostatic for intracellular brucellae. A combination of 5 to 50 mug/ml of streptomycin, which alone did not prevent the multiplication of intracellular brucellae, and 50 mug/ml of penicillin was bactericidal. Tetracycline at 0.5 to 1.0 mug/ml, which permitted growth or was bacteriostatic for brucellae within tissue cells, proved markedly bactericidal when combined with 10 mug/ml of streptomycin. Within certain limits, the synergistic effect of streptomycin and tetracycline did not appear to be a function of concentration. When the addition of streptomycin was delayed until 24 hr after the addition of tetracycline, the antibiotics acted synergistically on intracellular brucellae. To achieve intracellular synergism, five to ten times the effective extracellular concentration of streptomycin was required. Penicillin and tetracycline acted at the same concentration on extracellular and intracellular brucellae. These results suggest that tissue cell systems may prove useful for study of multiple drug therapy to be used in vivo against intracellular bacteria.

Effects of brucellaphage on extracellular and intracellular Brucella abortus.

SummaryStudies on the interactions between brucellaphage and Br. abortus, either under extracellular conditions or when the bacteria resided intracellularly within guinea pig mononuclear phagocytes maintained in vitro, showed: 1) inhibitory effects of untreated or UV-treated phage on the multiplication of brucellae, and 2) a lack of such effects in the case of heat-treated phage. These findings suggest an association between brucellacidal, or possibly brucellastatic, effects and phage protein. It was demonstrated that the effects on intracellular bacteria can occur even when exposure to phage is delayed until 2 hours after phagocytosis of the bacteria.