Development of a Brucella suis specific hybridisation probe and PCR which distinguishes B. suis from Brucella abortus

A genomic library was prepared from Brucella suis DNA (MboI digested) and cloned into the BamHI site of pUC18. Colony hybridisation using a probe prepared from purified B. suis DNA labelled with α 32P was carried out to identify colonies of interest. About 20 colonies, which gave an intense signal upon hybridisation with whole B. suis genomic DNA as a probe, were selected. Because of the high degree of DNA homology between B. suis and Brucella abortus, a short probe was chosen as it would more likely give species specificity. Of seven fragments selected to probe whole B. suis, B. abortus, and Yersinia enterocolitica DNA, one was found to hybridise with B. suis only. The probe was sequenced in two directions and sense and anti sense primers of 25 bp in length were chosen to yield a product of 421 bp. After optimisation of the PCR, a product of 420 bp was obtained with B. suis template DNA and two bands of 420 and 650 bp were detected with B. abortus template DNA. This is the first reported PCR of the Brucella genome where a single pair of primers will discriminate between B. suis and B. abortus. No band was observed when the two primers were used to amplify E. coli, Y. enterocolitica, Enterobacter cloacae, Staphylococcus aureus, Streptococcus uberis, Corynebacterium bovis, or Serratia marcescens template DNA

3D printed PLGA scaffold with nano-hydroxyapatite carrying linezolid for treatment of infected bone defects

Background: Linezolid has been reported to protect against chronic bone and joint infection. In this study, linezolid was loaded into the 3D printed poly (lactic-co-glycolic acid) (PLGA) scaffold with nano-hydroxyapatite (HA) to explore the effect of this composite scaffold on infected bone defect (IBD). Methods: PLGA scaffolds were produced using the 3D printing method. Drug release of linezolid was analyzed by elution and high-performance liquid chromatography assay. PLGA, PLGA-HA, and linezolid-loaded PLGA-HA scaffolds, were implanted into the defect site of a rabbit radius defect model. Micro-CT, H&E, and Masson staining, and immunohistochemistry were performed to analyze bone infection and bone healing. Evaluation of viable bacteria was performed. The cytocompatibility of 3D-printed composite scaffolds in vitro was detected using human bone marrow mesenchymal stem cells (BMSCs). Long-term safety of the scaffolds in rabbits was evaluated. Results: The linezolid-loaded PLGA-HA scaffolds exhibited a sustained release of linezolid and showed significant antibacterial effects. In the IBD rabbit models implanted with the scaffolds, the linezolid-loaded PLGA-HA scaffolds promoted bone healing and attenuated bone infection. The PLGA-HA scaffolds carrying linezolid upregulated the expression of osteogenic genes including collagen I, runt-related transcription factor 2, and osteocalcin. The linezolid-loaded PLGA-HA scaffolds promoted the proliferation and osteogenesis of BMSCs in vitro via the PI3K/AKT pathway. Moreover, the rabbits implanted with the linezolid-loaded scaffolds showed normal biochemical profiles and normal histology, which suggested the safety of the linezolid-loaded scaffolds. Conclusion: Overall, the linezolid-loaded PLGA-HA scaffolds fabricated by 3D printing exerts significant bone repair and anti-infection effects