The host response to bacterial bone infection involves a local upregulation of several acute phase proteins.

Bone infections often become chronic and can be difficult to diagnose. In the present study, the osseous gene expression of several acute phase proteins (APPs) during osteomyelitis was investigated in a porcine model of implant associated osteomyelitis (IAO) (sampled 5, 10 and 15 days after infection) and in slaughter pigs with spontaneous hematogenous osteomyelitis, and compared to gene expression in liver tissue. Furthermore, immunohistochemical (IHC) staining of the APP complement component C3 (C3) was performed on the porcine osteomyelitis lesions together with material from human patients with chronic osteomyelitis. In the porcine bone samples a local upregulation of the expression of several APP genes, including serum amyloid A (SAA) and C3, was observed during infection. In the liver, only C-reactive protein (CRP) and Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 were significantly upregulated. Serum concentrations of CRP, SAA and haptoglobin were only upregulated at day 5 in infected animals of the IAO model. This indicates a limited systemic response to osteomyelitis. Similar numbers of positive IHC stained C3 leukocytes were found in human and porcine bone samples with chronic osteomyelitis, indicating a high transcriptional value of porcine models of osteomyelitis. The local upregulation of APPs could potentially be used for diagnosing osteomyelitis.

The inflammatory response to bone infection - a review based on animal models and human patients.

Bone infections are difficult to diagnose and treat, especially when a prosthetic joint replacement or implant is involved. Bone loss is a major complication of osteomyelitis, but the mechanism behind has mainly been investigated in cell cultures and has not been confirmed in human settings. Inflammation is important in initiating an appropriate immune response to invading pathogens. However, many of the signaling molecules used by the immune system can also modulate bone remodeling and contribute to bone resorption during osteomyelitis. Our current knowledge of the inflammatory response relies heavily on animal models as research based on human samples is scarce. Staphylococcus aureus is one of the most common causes of bone infections and is the pathogen of choice in animal models. The regulation of inflammatory genes during prosthetic joint infections and implant-associated osteomyelitis has only been studied in rodent models. It is important to consider the validity of an animal model when results are extrapolated to humans, and both bone composition and the immune system of pigs has been shown to be more similar to humans, than to rodents. Here in vivo studies on the inflammatory response to prosthetic joint infections and implant-associated osteomyelitis are reviewed.

Pigs are useful for the molecular study of bone inflammation and regeneration in humans.

Pigs are used with increased frequency to model different kinds of orthopedic surgical conditions. In order to show the full potential of porcine models in orthopedic research, it is therefore required to examine the expression of bone regulatory genes in pigs affected by orthopedic surgery and compare it to the expression in humans and mice as mice, are one of the most applied animal species in orthopedics today. In the present study, the local molecular response to drilling of a tibial implant cavity, and the subsequent insertion of a steel implant was examined in a porcine model. Pigs were euthanized five days after drilling of the bone. The molecular response of 73 different genes was analyzed using a high-throughput quantitative polymerase chain reaction platform and compared to histopathology. Histologically, it was found that bone remodeling was initiated on day 5 after surgery and was associated with upregulation of several genes involved in bone degradation and formation ( CTSK, ACP5, IBSP, RANK, RANKL and COL1A1). Interleukin-6 and several acute-phase proteins (C3, SAA and ITIH4) were significantly upregulated, indicating their importance in the initial process of healing and osseointegration. All tested bone morphogenic proteins (BMP2, -4 and -7) including their inhibitor noggin were also significantly upregulated. Surprisingly, vascular endothelial growth factor A was not found to be regulated five days after surgery while several other vascular growth factors (ANGPT1, ANGPT2 and PTN) were upregulated. The pig was found to be a useful model for elucidation of bone regulatory genes in humans.

Treatment with Cefotaxime Affects Expression of Conjugation Associated Proteins and Conjugation Transfer Frequency of an IncI1 Plasmid in Escherichia coli.

Horizontal gene transfer (HGT) is the major mechanism responsible for spread of antibiotic resistance. Antibiotic treatment has been suggested to promote HGT, either by directly affecting the conjugation process itself or by selecting for conjugations subsequent to DNA transfer. However, recent research suggests that the effect of antibiotic treatment on plasmid conjugation frequencies, and hence the spread of resistance plasmids, may have been overestimated. We addressed the question by quantifying transfer proteins and conjugation frequencies of a blaCTX-M-1 encoding IncI1 resistance plasmid in Escherichia coli MG1655 in the presence and absence of therapeutically relevant concentrations of cefotaxime (CTX). Analysis of the proteome by iTRAQ labeling and liquid chromatography tandem mass spectrometry revealed that Tra proteins were significantly up-regulated in the presence of CTX. The up-regulation of the transfer machinery was confirmed at the transcriptional level for five selected genes. The CTX treatment did not cause induction of the SOS-response as revealed by absence of significantly regulated SOS associated proteins in the proteome and no significant up-regulation of recA and sfiA genes. The frequency of plasmid conjugation, measured in an antibiotic free environment, increased significantly when the donor was pre-grown in broth containing CTX compared to growth without this drug, regardless of whether blaCTX-M-1 was located on the plasmid or in trans on the chromosome. The results shows that antibiotic treatment can affect expression of a plasmid conjugation machinery and subsequent DNA transfer.

Bacterial Survival in Dictyostelium.

We performed an assay to test the ability of different E. coli strains to survive inside amoebal cells after ingestion. In the assay we incubated bacteria together with cells of Dictyostelium discoideum for six hours. After co-incubation most of the uningested bacteria were removed by centrifugation and the remaining uningested bacteria were killed by gentamicin. Gentamicin is used because it does not penetrate into eukaryotic cells allowing the ingested bacteria to survive the antibiotic treatment, whereas bacteria outside the amoebal cells are killed.

A role for copper in protozoan grazing - two billion years selecting for bacterial copper resistance.

The Great Oxidation Event resulted in integration of soft metals in a wide range of biochemical processes including, in our opinion, killing of bacteria by protozoa. Compared to pressure from anthropologic copper contamination, little is known on impacts of protozoan predation on maintenance of copper resistance determinants in bacteria. To evaluate the role of copper and other soft metals in predatory mechanisms of protozoa, we examined survival of bacteria mutated in different transition metal efflux or uptake systems in the social amoeba Dictyostelium discoideum. Our data demonstrated a strong correlation between the presence of copper/zinc efflux as well as iron/manganese uptake, and bacterial survival in amoebae. The growth of protozoa, in turn, was dependent on bacterial copper sensitivity. The phagocytosis of bacteria induced upregulation of Dictyostelium genes encoding the copper uptake transporter p80 and a triad of Cu(I)-translocating PIB -type ATPases. Accumulated Cu(I) in Dictyostelium was monitored using a copper biosensor bacterial strain. Altogether, our data demonstrate that Cu(I) is ultimately involved in protozoan predation of bacteria, supporting our hypothesis that protozoan grazing selected for the presence of copper resistance determinants for about two billion years.

Survival in amoeba--a major selection pressure on the presence of bacterial copper and zinc resistance determinants? Identification of a "copper pathogenicity island".

The presence of metal resistance determinants in bacteria usually is attributed to geological or anthropogenic metal contamination in different environments or associated with the use of antimicrobial metals in human healthcare or in agriculture. While this is certainly true, we hypothesize that protozoan predation and macrophage killing are also responsible for selection of copper/zinc resistance genes in bacteria. In this review, we outline evidence supporting this hypothesis, as well as highlight the correlation between metal resistance and pathogenicity in bacteria. In addition, we introduce and characterize the "copper pathogenicity island" identified in Escherichia coli and Salmonella strains isolated from copper- and zinc-fed Danish pigs.

Genome Sequences of Two Copper-Resistant Escherichia coli Strains Isolated from Copper-Fed Pigs.

The draft genome sequences of two copper-resistant Escherichia coli strains were determined. These had been isolated from copper-fed pigs and contained additional putative operons conferring copper and other metal and metalloid resistances.