A porcine model was used to examine the potential of human and porcine Staphylococcus aureus isolates to induce haematogenously spread osteomyelitis. Pigs were inoculated in the right femoral artery with one of the following S. aureus strains: S54F9 (from a porcine lung abscess; n = 3 animals), NCTC-8325-4 (a laboratory strain of human origin; n = 3 animals) and UAMS-1 (a human osteomyelitis isolate; n = 3 animals). Two pigs were sham inoculated with saline. At 11 or 15 days post infection the animals were scanned by computed tomography before being killed and subjected to necropsy examination. Osteomyelitis lesions were present in the right hind limb of all pigs inoculated with strain S54F9 and in one pig inoculated with strain NCTC-8325-4. Microscopically, there was extensive loss of bone tissue with surrounding granulation tissue. Sequestrated bone trabeculae were intermingled with colonies of S. aureus as demonstrated immunohistochemically. By peptide nucleic acid fluorescence in situ hybridization bacterial aggregates were demonstrated to be embedded in an opaque matrix, indicating that the bacteria had formed a biofilm. Development of experimental osteomyelitis was therefore dependent on the strain of bacteria inoculated and on the formation of a biofilm.

It is suggested that cyclooxygenase 2 (COX-2) derived prostaglandins contributes to the progressive bone loss seen in osteomyelitis lesions. In the present study we examined the expression of COX-2 in bones from 23 pigs with experimental osteomyelitis. Osteomyelitis was induced with Staphylococcus aureus and groups of animals were euthanized following 6h, 12h, 24h, 2 days, 5 days, 11 days and 15 days, respectively. Expression of COX-2 was evaluated immunohistochemically and combined with characterization of morphological changes in bone tissue. Furthermore, the serum concentrations of alkaline phosphatase and haptoglobin were measured. Extensive COX-2 expression by osteoblasts was present 2 days after inoculation together with many activated osteoclasts. Simultaneously, the serum concentration of alkaline phosphatase decreased whereas the haptoglobin concentration increased. This is the first in vivo study showing an early wave of COX-2 mediated bone resorption during osteomyelitis. Therefore, treatment aiming to reduce the break down of bone tissue directed by the COX-2 pathway might be suggested early in the course of the disease.

Spx is a global regulator that is widespread among the low G+C Gram-positive bacteria. Spx has been extensively studied in Bacillus subtilis, where it acts as an activator and a repressor of transcription in response to disulfide stress. Under non-stress conditions, Spx is rapidly degraded by the ClpXP protease. This degradation is enhanced by the YjbH adaptor protein. Upon disulfide stress, the amount of Spx rapidly increases due to a decrease in degradation. In the opportunistic pathogen Staphylococcus aureus, Spx is a global regulator influencing growth, biofilm formation and general stress protection, and cells lacking the spx gene exhibit poor growth also under non-stress conditions. To investigate the mechanism by which the activity of Spx is regulated we identified a homolog in S. aureus of the B. subtilis yjbH gene. The gene encodes a protein that shows approximately 30% sequence identity to YjbH of B. subtilis. Heterologous expression of S. aureus yjbH in a B. subtilis yjbH mutant restored Spx to wild type levels both under non-stress conditions and under conditions of disulfide stress. From these studies we conclude that the two YjbH homologues have a conserved physiological function. Accordingly, inactivation of yjbH in S. aureus increased the level of Spx protein and transcription of the Spx-regulated gene trxB. Notably, the yjbH mutant exhibited reduced growth and increased pigmentation, and both phenotypes were reversed by complementation of the yjbH gene.

In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here re-assessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.

Strains from genus Gallibacterium colonize and occasionally cause disease in a range of bird species. The seven species identified vary with respect to haemolytic activity: Gallibacterium genomospecies 1 and 2 are haemolytic, while G. anatis comprise both haemolytic and nonhaemolytic strains. The remaining species are all non-haemolytic. We previously reported that G. anatis strain 12656-12 expresses an atypical RTX-toxin (repeat in toxin), GtxA, responsible for the haemolytic activity and likely to be a major virulence factor. The aim of this study was to investigate the basis of the variation in haemolytic activity observed among Gallibacterium species and strains. Using PCR and dot blotting we found that the gtxA gene was absent from non-haemolytic Gallibacterium species, but present in Gallibacterium genomospecies 1 and 2. Surprisingly, gtxA was present in both haemolytic and non-haemolytic strains of G. anatis. However, in two out of seven of the non-haemolytic G. anatis strains, gtxA was interrupted by an insertion sequence. We identified a new type I secretion system locus (gtxEBD) and showed that this locus is required for export of GtxA. The gtxEBD locus was identified in all strains possessing gtxA, thus, lack of export system genes cannot explain the non-haemolytic phenotype. Instead we examined expression of gtxA at both the transcript- and protein level by Northern- and Western blotting and found that expression of gtxA varied significantly between strains. In conclusion, we have shown that differences in haemolytic properties among strains of Gallibacterium may be explained by both genotypic differences and by differential expression.

A porcine model of acute, haematogenous, localized osteomyelitis was established. Serial dilutions of Staphylococcus aureus [5-50-500-5000-50 000 CFU/kg body weight (BW) suspended in saline or saline alone] were inoculated into the right brachial artery of pigs (BW 15 kg) separated into six groups of two animals. During the infection, blood was collected for cultivation, and after the animals were killed from day 5 to 15, they were necropsied and tissues were sampled for histopathology. Animals receiving ≤500 CFU/kg BW were free of lesions. Pigs inoculated with 5000 and 50 000 CFU/kg BW only developed microabscesses in bones of the infected legs. In the centre of microabscesses, S. aureus was regularly demonstrated together with necrotic neutrophils. Often, bone lesions resulted in trabecular osteonecrosis. The present localized model of acute haematogenous osteomyelitis revealed a pattern of development and presence of lesions similar to the situation in children. Therefore, this model should be reliably applied in studies of this disease with respect to e.g. pathophysiology and pathomorphology. Moreover, because of the regional containment of the infection to a defined number of bones, the model should be applicable also for screening of new therapy strategies.

In recent years, small RNAs (sRNAs) have been identified as important regulators of gene expression in bacteria. Most sRNAs are encoded from intergenic regions and are only expressed under highly specific growth conditions. In Staphylococcus aureus, the alternative sigma factor, σ(B), is known to contribute to the overall stress response, antibiotic resistance, and virulence. The σ(B) regulon in S. aureus is well described and comprises approximately 200 annotated genes, including several genes encoding virulence factors. In the present study, we have identified three novel σ(B)-dependent transcripts encoded from genomic regions previously annotated as intergenic. All three transcripts, named SbrA, SbrB, and SbrC, are highly conserved in S. aureus, and we confirmed their presence in four different isolates (SH1000, Newman, COL, and UAMS-1). Curiously, two of these genes (sbrA and sbrB) were found to contain open reading frames encoding small, highly basic peptides that are conserved among Staphylococci. The third transcript (SbrC) did not contain any likely open reading frame and thus constitute a genuine non-coding sRNA. The functions of these genes are currently unknown but are likely to be important for the σ(B)-mediated response of S. aureus to adverse conditions.

Gallibacterium anatis is a pathogen in chickens and other avian species where it is a significant cause of salpingitis and peritonitis. We found that bacterial cells and cell-free, filter-sterilised culture supernatant from the haemolytic G. anatis biovar haemolytica were highly cytotoxic towards avian-derived macrophage-like cells (HD11). We obtained the genome sequence of G. anatis 12656-12 and used a rational approach to identify a gene predicted to encode a 2026 amino acid RTX-toxin, which we named GtxA (Gallibacterium toxin). The construction of a gtxA knock-out mutant showed gtxA to be responsible for G. anatis haemolytic and leukotoxic activity. In addition, Escherichia coli expressing gtxA and an adjacent acyltransferase, gtxC, became cytolytic. GtxA was expressed during in vitro growth and was localised in the extracellular protein fraction in a growth phase dependent manner. GtxA had an unusual modular structure; the C-terminal 1000 amino acids of GtxA were homologous to the classical pore-forming RTX-toxins in other members of Pasteurellaceae. In contrast, the N-terminal approximately 950 amino acids had few significant matches in sequence databases. Expression of truncated GtxA proteins demonstrated that the C-terminal RTX-domain had a lower haemolytic activity than the full-length toxin, indicating that the N-terminal domain was required for maximal haemolytic activity. Cytotoxicity towards HD11 cells was not detected with the C-terminal alone, suggesting that the N-terminal domain plays a critical role for the leukotoxicity.

We present a simple assay to examine effects of compounds on virulence gene expression in the human pathogen, Staphylococcus aureus. The assay employs transcriptional reporter strains carrying lacZ fused to central virulence genes. Compounds affecting virulence gene expression and activity of the agr locus are scored as colour change in presence of a chromogenic beta-galactosidase substrate. The assay can be used to screen for novel anti-virulence compounds from many different sources such as fungi as demonstrated here.

Current models for global virulence regulation in Staphylococcus aureus are mainly based on studies performed with only a limited number of laboratory strains derived from NCTC8325. In these strains the small regulatory RNA, RNAIII, has a central role in virulence gene regulation. Recently, RNAIII was suggested to control transcription of target genes partly by inhibiting translation of the transcriptional regulator Rot. The present study was undertaken to examine if the model for RNAIII/Rot-dependent virulence regulation is conserved among clinical strains. To this end, we used Rot antibodies to directly assess the amount of Rot protein in 4 well-characterized S. aureus laboratory strains (8325-4, COL, Newman, and UAMS-1) and in 9 strains of clinical origin (encompassing USA300 and Mu50). Additionally, the cellular amount of RNAIII and rot mRNA was determined in all strains. The experiments revealed considerable variation in the Rot and RNAIII levels between strains. However, in the majority of strains the cellular amount of Rot was inversely correlated to the RNAIII level. As we demonstrate that Rot is a stable protein and that the level of rot transcript appeared similar in all strains, our data support that the model for RNAIII-mediated inhibition of rot mRNA translation is conserved among clinical strains. Assessment of Rot-dependent regulation of target genes revealed that Rot is a positive regulator of spa (protein A) transcription in all strains examined. In contrast, Rot repression of sspA (serine protease) and hlb (beta-hemolysin) transcription was not conserved between strains. From this study, we conclude that while the paradigm for understanding RNAIII-dependent regulation of Rot is well-conserved, regulation of single genes is subject to considerable strain variation. We propose that variation in global regulatory networks contribute considerably to the phenotypic variation observed between S. aureus isolates.