The objective of this study was to set up anin vivogentamicin susceptibility test for biofilm prevention in bone tissue and on implants. Twenty-five pigs were allocated to six groups. Pigs in group A (n= 6) were inoculated with saline. Pigs in groups B (n= 6), C (n= 3), D (n= 3), E (n= 3), and F (n= 4) were inoculated with 10 μl saline containing 104CFU ofStaphylococcus aureus. Different concentrations based on the MIC of gentamicin for the specific strain were added to the 10-μl inoculum for groups C (160× MIC), D (1,600× MIC), E (16,000× MIC), and F (160,000× MIC). The inocula were injected into a predrilled tibial implant cavity, followed by insertion of a steel implant (2 by 15 mm). The pigs were euthanized after 5 days.In vitro, all the doses used were found to be bactericidal after up to 6 h. All implant cavities of pigs inoculated with bacteria and bacteria plus 160× MIC or 1,600× MIC of gentamicin were positive forS. aureus. In animals in each of groups E (16,000× MIC) and F (160,000× MIC), 2/3 and 1/4 of the implant cavities wereS. aureuspositive, respectively. By grouping groups C and D (<10,000× MIC) and groups E and F (>10,000× MIC), a significant decrease in the number of implant-attached bacteria was seen only between the high-MIC-value group and group B. Histologically, it was demonstrated that 1,600×, 16,000×, and 160,000× MIC resulted in a peri-implant tissue reaction comparable to that in saline-inoculated animals.In vivo, the antimicrobial tolerance of the inoculated planktonic bacteria was increased byin vivo-specific factors of acute inflammation. This resulted in bacterial aggregation and biofilm formation, which further increased the gentamicin tolerance. Thus, susceptibility patternsin vitromight not reflect the actualin vivosusceptibility locally within a developing infectious area.

The assessment of the age of bruises inflicted on livestock is an important component of veterinary forensic pathology investigations. However, the sampling site within a bruise, the anatomical location and the mass and speed of the object inflicting the blunt trauma might influence the intensity of the inflammatory reaction. In the present study, the variation of the inflammatory reaction within and along experimental porcine bruises was evaluated in order to determine the optimal sampling site. Moreover, we evaluated if a combination of histological characteristics and gene expression signatures was able to differentiate bruises according to anatomical location, age of bruises and the speed and mass of the object used to cause the impact. Twelve experimental slaughter pigs were anesthetized, and on each animal four blunt traumas were inflicted on the back using either a plastic tube or an iron bar, respectively. The pigs were euthanized at 2, 5 or 8 h after infliction. Following gross examination, skin and underlying muscle tissue were sampled from the center and both ends of bruises and evaluated histologically. Subcutaneous fat tissue from the center of the bruises was sampled for quantitative real-time polymerase chain reaction to evaluate mRNA expression of 13 selected genes. Uninjured tissue was sampled from the right thigh of all pigs and served as control tissue. The amount of tissue damage and the intensity of the inflammatory reaction in bruises depended on the sampling site within and along a bruise, the anatomical location and the age of the bruise. The optimal site for sampling, i.e. the most pronounced inflammatory reaction, was at the center of the bruises where the plastic tube or iron bar first struck the skin. Moreover, bruises inflicted in areas with a thin layer of subcutaneous fat tissue showed more damage and inflammation in the underlying muscle tissue compared to bruises inflicted in areas with a thicker layer of subcutaneous fat tissue. In addition, hemorrhage in the muscle tissue was more likely present when bruises were inflicted with an iron bar compared to a plastic tube. Combining histology and mRNA expression of the 13 genes showed that the age of bruises could be determined with a precision of ±2.04 h. Moreover, the age of bruises could be determined with a precision of ±1.84 h based solely on mRNA expression of a selection of four genes.

Aim: Visualization of Staphylococcus aureus biofilm using histochemical staining and combined histochemistry (HC) and immunohistochemistry (IHC).Methods: The ability of S. aureus S54F9 to form biofilm was tested in vitro. Hereafter, infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with S. aureus strain S54F9. The infection time was five and fifteen days, respectively. Twenty-five different histochemical staining protocols were tested in order to find the stains that could identify extracellular biofilm matrix. Protocols with an optimal visualization of biofilm extracellular matrix were combined with an immunohistochemical protocol based on a specific antibody against S. aureus. The combined protocols were applied to the tissue from the porcine models and to infected bone tissue from a child suffering from chronic staphylococcal osteomyelitis for more than a year.Results: S. aureus S54F9 showed an ability to form biofilm in vitro. Visualization of biofilm, i.e. bacterial cells and extracellular matrix in different colours, was seen when the immunohistochemical protocol was combined with Alcian Blue pH3, Luna and Methyl-pyronin green. The bacterial cells were red to light brown and the extracellular matrix either light blue, blue or orange depending on the histochemical stain. In the porcine models and the human case 10 and 90 percent, respectively, of the bacterial aggregates in a 100x magnification field displayed both the extracellular matrix and the bacterial cells simultaneously in two different colours.Conclusions: A combination of HC and IHC can be used to diagnose and characterise biofilm infections on a routine basis.

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Deficient levels of milk osteopontin (OPN) in infant formula may partly account for developmental differences between infants fed formula or maternal milk. We hypothesized that a milk diet supplemented with bovine milk OPN improves gut, immunity and brain development and tested this in a preterm pig model. Preterm pigs delivered by cesarean section (90% gestation) were fed raw bovine milk (CON, n = 19) or the same diet supplemented with a physiologically relevant dose of OPN (46 mg/(kg·d), n = 16). Endpoints related to clinical outcomes, systemic immunity and neurocognitive development were assessed during the study and gut tissues were collected at Day 19. Growth pattern, early motor development and most systemic immune parameters were similar between OPN and CON pigs. The OPN pigs had higher villus-to-crypt ratios than CON pigs and higher monocyte and lymphocyte counts on Day 8. Gut digestive and absorptive functions and cognitive performance (T-maze test) were similar between OPN and CON pigs. In conclusion, dietary supplementation with OPN above basal bovine milk levels induced minor improvements in gut structure and systemic immunity without any effects on cognitive performance. The minimal levels of OPN in infant formula to secure optimal adaptation in the immediate neonatal period remain to be determined.