Background Xenorhabdus spp. live in close symbiosis with nematodes of the Steinernema genus. Steinernema nematodes infect an insect larva and release their symbionts into the haemocoel of the insect. Once released into the haemocoel, the bacteria produce bioactive compounds to create a semi-exclusive environment by inhibiting the growth of bacteria, yeasts and molds. The antimicrobial compounds thus far identified are xenocoumacins, xenortides, xenorhabdins, indole derivatives, xenoamicins, bicornutin and a number of antimicrobial peptides. The latter may be linear peptides such as the bacteriocins xenocin and xenorhabdicin, rhabdopeptides and cabanillasin, or cyclic, such as PAX lipopeptides, taxlllaids, xenobactin and szentiamide. Thus far, production of antimicrobial compounds have been reported for Xenorhabdus nematophila, Xenorhabdus budapestensis, Xenorhabdus cabanillasii , Xenorhabdus kozodoii , Xenorhabdus szentirmaii , Xenorhabdus doucetiae , Xenorhabdus mauleonii , Xenorhabdus indica and Xenorhabdus bovienii . Here we describe, for the first time, PAX lipopeptides and xenocoumacin 2 produced by Xenorhabdus khoisanae . These compounds were identified using ultraperformance liquid chromatography, linked to high resolution electrospray ionisation mass spectrometry and tandem mass spectrometry. Results Cell-free supernatants of X. khoisanae SB10 were heat stable and active against Bacillus subtilis subsp. subtilis , Escherichia coli and Candida albicans . Five lysine-rich lipopeptides from the PAX group were identified in HPLC fractions, with PAX1’ and PAX7 present in the highest concentrations. Three novel PAX7 peptides with putative enoyl modifications and two linear analogues of PAX1’ were also detected. A small antibiotic compound, yellow in colour and λ max of 314 nm, was recovered from the HPLC fractions and identified as xenocoumacin 2. The PAX lipopeptides and xenocoumacin 2 correlated with the genes and gene clusters in the genome of X. khoisanae SB10. Conclusion With UPLC-MS and MS e analyses of compounds in the antimicrobial complex of X. khoisanae SB10, a number of PAX peptides and a xenocoumacin were identified. The combination of pure PAX1’ peptide with xenocoumacin 2 resulted in high antimicrobial activity. Many of the fractions did, however, contain labile compounds and some fractions were difficult to resolve. It is thus possible that strain SB10 may produce more antimicrobial compounds than repor...

Periprosthetic joint infections (PJI), caused by methicillin-resistant Staphylococcus aureus (MRSA), is the major cause of total hip arthroplasty (THA) failures. Traditionally, MRSA is treated with vancomycin, administrated intravenously or applied directly onto infected tissue. The effect of direct (as opposed to systemic) vancomycin treatment on bone formation and remodelling is largely unknown. The minimal inhibitory concentration (MIC) of vancomycin was determined by adding 200 µl of different concentrations (1 -20 µg/ml) to actively growing cultures of S. aureus Xen 31 (methicillin-resistant) and S. aureus Xen 36 (methicillin-sensitive), respectively, and recording changes in optical density over 24 h. Bone marrow-derived and proximal femur-derived mesenchymal stem cells (bmMSCs and pfMSCs) from rat femora were exposed to 1 x MIC (5 µg/ml) and 4 x MIC (20 µg/ml) of vancomycin for 7 days. Cell viability was determined by staining with crystal violet and MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), respectively, and osteogenic differentiation by staining with Alizarin Red S. Vancomycin had no effect on the viability of bmMSCs and pfMSCs, even at high levels (20 µg/ml). The osteogenic differentiation of pfMSCs was partially inhibited, while osteogenesis in bmMSCs was not severely affected. The direct application of vancomycin to infected bone tissue, even at excessive levels, may preserve the viability of resident MSC populations. Short-term demineralization may thus be reversed after cessation of vancomycin treatment, improving the outcome of THA surgery.

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Species from the genus Xenorhabdus, endosymbiotic bacteria of Steinernema nematodes, produce several antibacterial and antifungal compounds, some of which are anti-parasitic. In this study, we report on the effect growth conditions have on the production of antimicrobial compounds produced by Xenorhabdus khoisanae J194. The strain was cultured in aerated and non-aerated broth, respectively, and on solid media. Production of antimicrobial compounds was detected after 24 h of growth in liquid media, with highest levels recorded after 96 h. Highest antimicrobial activity was obtained from cells cultured on solid media. By using ultraperformance liquid chromatography linked to mass spectrometry and HPLC, a plethora of known Xenorhabdus compounds were identified. These compounds are the PAX lipopeptides (PAX 1′, PAX 3′, PAX 5, and PAX 7E), xenocoumacins and xenoamicins. Differences observed in the MS-MS fractionation patterns collected in this study, when compared to previous studies indicated that this strain produces novel xenoamicins. Three novel antimicrobial compounds, khoicin, xenopep and rhabdin, were identified and structurally characterized based on MS-MS fractionation patterns, amino acid analysis and whole genome analysis. The various compounds produced under the three different conditions indicates that the secondary metabolism of X. khoisanae J194 may be regulated by oxygen, water activity or both. Based on these findings X. khoisanae J194 produce a variety of antimicrobial compounds that may have application in disease control.

Bacteria colonizing the surface of orthopedic implants are responsible for most postoperative periprosthetic joint infections. A possible alternative route for drug delivery is described in this study by utilizing the bulk of the implant itself as a reservoir. Drug release is enabled by manufacturing of integrated permeable structures possessing high porosity through application of selective laser melting technology. The concept was evaluated in two paths, with 400 μm permeable thin walls and with dense reservoirs containing an integrated 950 μm permeable wall. Components were designed and preprocessed as separate parts, allowing for allocation of different settings of laser power and scanning speed. Lowering the energy input into the selective laser melting process to induce intermittent melting of the Ti6Al4V ELI powder produced porous components through which vancomycin was released with differing profiles. Static water contact angle measurements demonstrated a significant effect on the hydrophilicity by permeable wall thickness. Relative porosities of the 400 μm structures were determined with microcomputed tomography analyses. A transition zone of 21.17% porosity was identified where release profiles change from porosity-dependent to near free diffusion. Antimicrobial activity of released vancomycin was confirmed through evaluation against Staphylococcus aureus Xen 36 in two separate agar diffusion assays. The approach is promising for incorporation into the design and manufacturing of next-generation prosthetic implants with controlled release of antibiotics in situ and the subsequent prevention of periprosthetic joint infections.