Microbiological and ultrastructural evaluation of bacteriophage 191219 against planktonic, intracellular and biofilm infection with Staphylococcus aureus.

Infections of orthopaedic implants, such as fracture fixation devices and total-joint prostheses, are devastating complications. Staphylococcus aureus (S. aureus) is a predominant pathogen causing orthopaedic-implant biofilm infections that can also internalise and persist in osteoblasts, thus resisting antibiotic therapy. Bacteriophages are a promising alternative treatment approach. However, data on the activity of bacteriophages against S. aureus, especially during intracellular growth, and against in vivo biofilm formation on metals are scarce. Therefore, the present study evaluated the in vitro efficacy of S. aureus bacteriophage 191219, alone as well as in combination with gentamicin and rifampicin, to eradicate S. aureus strains in their planktonic stage, during biofilm formation and after internalisation into osteoblasts. Further, the invertebrate model organism Galleria mellonella was used to assess the activity of the bacteriophage against S. aureus biofilm on metal implants with and without antibiotics. Results demonstrated the in vitro efficacy of bacteriophage 191219 against planktonic S. aureus. The phage was also effective against in vitro S. aureus biofilm formation in a dose-dependent manner and against S. aureus internalised in an osteoblastic cell line. Transmission electron microscopy (TEM) analysis showed bacteriophages on S. aureus inside the osteoblasts, with the destruction of the intracellular bacteria and formation of new bacteriophages. For the Galleria mellonella infection model, single administration of phage 191219 failed to show an improvement in survival rate but appeared to show a not statistically significant enhanced effect with gentamicin or rifampicin. In summary, bacteriophages could be a potential adjuvant treatment strategy for patients with implant-associated biofilm infections.

Vertebral osteomyelitis is characterised by increased RANK/OPG and RANKL/OPG expression ratios in vertebral bodies and intervertebral discs.

Vertebral osteomyelitis (VO) is an infection of the spine mainly caused by bacterial pathogens. The pathogenesis leading to destruction of intervertebral discs (IVDs) and adjacent vertebral bodies (VBs) is poorly described. The present study aimed at investigating the connection between infection and bone/disc metabolism in VO patients. 14 patients with VO (infection group) and 14 patients with burst fractures of the spine (fracture group; control) were included prospectively. Tissue biopsies from affected IVDs and adjacent VBs were analysed by RT-qPCR for mRNA-expression levels of 18 target genes including chemokines, adipokines and genes involved in bone metabolism. Most importantly, the receptor activator of NF-κB/osteoprotegerin (RANK/OPG) expression ratio was drastically elevated in both VBs and IVDs of the infection group. In parallel, expression of genes of the prostaglandin-E2-dependent prostanoid system was induced. Such genes regulate tissue degradation processes via the triad OPG/RANK/RANKL as well as via the chemokines IL-8 and CCL-20, whose expression was also found to be increased upon infection. The gene expression of the adipokine leptin, which promotes inflammatory tissue degradation, was higher in IVD tissue of the infection group, whereas the transcription of omentin and resistin genes, whose functions are largely unknown in the context of infectious diseases, was lower in infected VBs. In summary, similar expression patterns of pro-inflammatory cytokines and pro-osteoclastogenic factors were identified in VBs and IVDs of patients suffering from VO. This suggests that common immuno-metabolic pathways are involved in the mechanisms leading to tissue degradation in VBs and IVDs during VO.

Extensor tendon ruptures in rheumatoid wrists.

BACKGROUND AND AIMS: Rheumatoid arthritis is a chronic inflammatory disease. The associated involvement of hands and tendons is over 90% and impairs overall function. In the course of the disease, the joints are often operated on. During this operation, ruptures of the extensor tendons are found by chance without the patients noticing them. The aim of this retrospective study is the prevalence of extensor tendon rupture. Which tendon is destroyed most frequently? How can the functional outcome be measured after reconstruction? MATERIALS AND METHODS: From 1572 operations on rheumatoid wrists, 61 extensor tendon ruptures were identified in 41 patients. The average time between the first rheumatic symptoms of the hand and surgery was 6.4 years. The average duration of RA was 7.8 years. 26 patients with 27 tendon reconstructions were included in the follow-up with an average postoperative duration of 4.6 years (3 to 14.2 years). RESULTS: Extensor tendons ruptures typically occurred at mechanically stressed sites. The most frequent rupture was found in the extensor pollicis longus tendon (21 tendons), followed by the small finger extensor tendon (14 tendons). A transfer was performed on 7 tendons. Fifty-five tendon lesions were sutured at other intact tendons. Free grafts were not used. The results in Clayton and QuickDASH scores were significantly different. Functional improvement was consistent with the results of tendon reconstructions in healthy control groups. CONCLUSION: In rheumatoid patients, a rupture of an extensor tendon must be expected at 4%. Patients tolerate and compensate this damage for a long time. The function of the hand including the tendon function is the most important factor in assessing the success of the operation. The subjective patient acceptance depends on the progress of the underlying disease, postoperative care (ergotherapy, physiotherapy, orthosis) and the patients' demands.

Successful Total Elbow Replacement after Septic Arthritis with Staphylococcus aureus- a Case Report and Review of the Literature.

Septic arthritis of the elbow seems to be a contraindication for total elbow arthroplasty (TEA). We here describe a 65-year-old male, American Society of Anesthesiologists (ASA) class 3 - patient, with a severely destructed right elbow due to septic arthritis with Staphylococcus aureus. His treatment consisted of multiple irrigation and debridement procedures including resection of the distal humerus, soft tissue coverage by local rotational flap and the use of a gentamicin-vancomycin loaded PMMA spacer, i.v. and oral antibiotics. After eradication of infection, a constraint cemented TEA could then successfully be performed eight months after the initial surgery and twenty-five weeks after the last debridement procedure. Twenty-one months after the TEA, the patient remained infect free and shows excellent functional results: Disabilities of the Arm, Shoulder and Hand (DASH) score: 38.3, Broberg and Morrey score: 91/100, Mayo elbow score: 95/100. To the best of our knowledge this is the first case in the literature that demonstrates TEA after septic elbow arthritis with Staphylococcus aureus. Although TEA is known as a typical surgical procedure with a low volume in numbers and higher complication rates, such as elevated infection rates compared to other types of arthroplasty, septic arthritis with Staphylococcus aureuscan successfully be performed after eradication of the infection and targeted antibiotic therapy. Key words:total elbow arthroplasty, total elbow replacement, septic arthritis, Staphylococcus aureus.

Staphylococcus epidermidis saeR is an effector of anaerobic growth and a mediator of acute inflammation.

The saeRS two-component regulatory system regulates transcription of multiple virulence factors in Staphylococcus aureus. In the present study, we demonstrated that the saePQRS region in Staphylococcus epidermidis is transcriptionally regulated in a temporal manner and is arranged in a manner similar to that previously described for S. aureus. Studies using a mouse foreign body infection model demonstrated that the virulence of strain 1457 and the virulence of a mutant, strain 1457 saeR, were statistically equivalent. However, histological analyses suggested that the polymorphonuclear neutrophil response at 2 days postinfection was significantly greater in 1457-infected mice than in 1457 saeR-infected mice, demonstrating that SaeR influences the early, acute phases of infection. Microarray analysis demonstrated that a saeR mutation affected the transcription of 65 genes (37 genes were upregulated and 28 genes were downregulated); in particular, 8 genes that facilitate growth under anaerobic conditions were downregulated in 1457 saeR. Analysis of growth under anaerobic conditions demonstrated that 1457 saeR had a decreased growth rate compared to 1457. Further metabolic experiments demonstrated that 1457 saeR had a reduced capacity to utilize nitrate as a terminal electron acceptor and exhibited increased production of lactic acid in comparison to 1457. These data suggest that in S. epidermidis SaeR functions to regulate the transition between aerobic growth and anaerobic growth. In addition, when grown anaerobically, 1457 saeR appeared to compensate for the redox imbalance created by the lack of electron transport-mediated oxidation of NADH to NAD+ by increasing lactate dehydrogenase activity and the subsequent oxidation of NADH.

The presence of icaADBC is detrimental to the colonization of human skin by Staphylococcus epidermidis.

Previous studies have demonstrated that Staphylococcus epidermidis isolates colonizing the skin of healthy humans do not typically encode icaADBC, the genes responsible for the production of polysaccharide intercellular adhesin or biofilms. It was therefore hypothesized that the presence of icaADBC was deleterious to the successful colonization of human skin by S. epidermidis. Using a human skin competition model, it was determined that the strong biofilm-producing S. epidermidis strain 1457 was outcompeted at 1, 3, and 10 days by an isogenic icaADBC mutant (1457 ica::dhfr), suggesting a fitness cost for carriage of icaADBC.

FOXO3-mediated chemo-protection in high-stage neuroblastoma depends on wild-type TP53 and SESN3.

Forkhead box O class transcription factors are homeostasis regulators that control cell death, longevity and therapy-resistance. In neuroblastoma (NB), nuclear FOXO3 correlates with stage M disease and poor prognosis. To analyze whether FOXO3 contributes to drug-resistance in this childhood cancer, we investigated how different high-stage-derived NB cells respond to the activation of an ectopic FOXO3 allele. We found endogenous FOXO3 mostly localized to the nucleus-upon activation of an ectopic, 4OHT-activated FOXO3(A3)ER fusion protein two of the cell lines underwent apoptosis, whereas in the others FOXO3-activation even increased survival during drug-treatment. In the latter cell type, FOXO3 did not induce the BH3-only protein BCL2L11/BIM due to impaired binding of FOXO3 to the BIM-promoter, but still activated other FOXO3 targets. It was shown before that FOXO3 and TP53 physically interact with each other at two different regions-the TP53-N-terminus binds to the FOXO3-DNA binding domain (DBD) and the FOXO3-C-terminus interacts with the TP53-DBD. Interestingly, cell lines that undergo FOXO3-induced cell death carry homozygous point mutations in the TP53-DBD near the structural hotspot-mutation-site R175H, which abrogated FOXO3-TP53 interaction. In contrast, in FOXO3-death-resistant cells no point mutations in the TP53-DBD were found-in these cells FOXO3-TP53 complexes are formed and FOXO3-binding to the BIM-promoter, but not the induction of the detoxifying protein SESN3, were prevented, which in turn increased chemo-protection in this type of high-stage-derived NB cells. Our combined data suggest that FOXO3 steps in as a death inducer in case of TP53-mutation, whereas functional TP53 alters FOXO3-target-promoter-recognition, which prevents death induction by FOXO3 and instead increases chemo-protection and survival of NB cells. This novel mechanism may explain the low incidence of TP53 mutation in high-stage NB at diagnosis and suggests FOXO3 as a therapeutic target for this childhood malignancy.

Initial characterizations of nontransformed and transformed [3H ]aldosterone-type I receptor complexes in brain cytosol.

Incubation of [3H]aldosterone-type I receptor complexes in mouse brain cytosol with the chaotropic anion thiocyanate increased the fraction of receptors retained by DNA-cellulose from less than 10% to over 40%, whereas it decreased the fraction retained by protamine sulfate from more than 90% to less than 10%. Thiocyanate-induced transformation to the DNA-binding species was also accompanied by a 2.1-fold decrease in the rate of [3H]aldosterone dissociation from type I receptors as well as by an increase in the apparent positive charge and hydrophobicity of the surface of these receptors, as revealed by DEAE Bio-Gel ion exchange and pentyl agarose hydrophobic interaction chromatography. Sucrose density gradient sedimentation and Sephacryl S-300 gel exclusion chromatography revealed a reduction in the sedimentation coefficient and Stokes radius of the steroid-receptor complex from 9.6S and 8.0 nm before to 4.7S and 6.1 nm after transformation, respectively. These changes in hydrodynamic parameters were found to correspond to a 2.8-fold reduction in the apparent molecular mass from 331,000 before to 120,000 after transformation. In view of these various findings as well as the known differential affinity of protamine sulfate for the 90K heat shock protein, we suggest that thiocyanate-induced transformation is initiated by the dissociation of two molecules of heat shock protein from each steroid/DNA-binding type I receptor subunit.

Aldosterone-stimulated down-regulation of both type I and type II adrenocorticosteroid receptors in mouse brain is mediated via type I receptors.

The concentrations of type I and type II adrenocorticosteroid receptors in brain cytosol obtained from adrenalectomized-ovariectomized female mice were measured with five different assay conditions. Among the five brain regions studied, hippocampus had the highest concentration of type I receptors, whereas cerebral cortex had the highest concentration of type II receptors. The value of properly correcting for dexamethasone cross-binding to type I receptors when type II receptors are being assayed was demonstrated using the type II receptor-selective ligand RU28362. A time-course study revealed a transient up-regulation of both receptor classes in most brain regions after adrenalectomy-ovariectomy, with maximal values achieved 3-5 days postsurgery and a reduction to near-intact levels by 16 days postsurgery. A single sc injection of aldosterone given to adrenalectomized-ovariectomized mice produced a profound down-regulation of type I receptors in hippocampal, cerebral cortex, hypothalamic, brain stem, and cerebellar samples, whereas it down-regulated type II receptors only in hippocampal and cerebral cortical samples. A similar injection of RU28362 failed to down-regulate type I receptors in any brain region, but it did reduce the concentration of type II receptors in all brain regions except cerebellum. The actions of aldosterone appear to be mediated solely through type I receptors, since injections of the type I receptor antagonist RU26752 prevented aldosterone-induced down-regulation of both type I and type II receptors, whereas RU26752 had no effect on the down-regulatory actions of RU28362. The ability of aldosterone to down-regulate type I, but not type II, receptors in hypothalamic, brain stem, and cerebellar samples suggests that type I and type II receptors are concentrated in separate populations of cells in these brain regions, whereas in hippocampus and cerebral cortex there is a sufficient degree of colocalization to permit type II receptor down-regulation via the action of aldosterone-type I receptor complexes. We speculate that this action is mediated at least in part at the genomic level by the suppression of type I and type II receptor mRNA synthesis brought about by the interactions of transformed aldosterone-type I receptor complexes with the DNA regulatory elements upstream from the genes for these receptors.

High affinity binding and regulatory actions of dexamethasone-type I receptor complexes in mouse brain.

It is often implied that the various molecular, physiological, and behavioral responses to the glucocorticoid dexamethasone (DEX) are mediated in brain exclusively via the interactions of this synthetic steroid with the classical glucocorticoid (type II) receptor. The results reported in this study, however, suggest this generalization may, at least for the female mouse, be too restrictive. In the first experiment we compared the efficacy of the mineralocorticoid aldosterone (ALDO) with that of DEX to measure the classical mineralocorticoid (type I) receptor in brain cytosol. Since both of these steroids also bind to type II receptors, our assays included the type II receptor-selective ligand, RU26988. Whereas the specific binding of ALDO to type I receptors was largely unaffected by a 10-fold increase in the concentration of RU26988 (50- vs. 500-fold excess), there was a dramatic reduction in the specific binding of DEX. In a follow-up experiment, Scatchard analyses were used to confirm the differential affinity of RU26988 for DEX- vs. ALDO-type I receptor-binding sites and to reveal that the affinity of type I receptors for DEX (Kd approximately 0.83 nM) was nearly as high as it was for ALDO (Kd approximately 0.46 nM). A series of competition studies indicated that the competitive affinity (Kdc) of DEX for the ALDO-binding site was equivalent to the Kd computed in the saturation analyses, thus suggesting that the high affinity binding sites for DEX and ALDO on type I receptors may be equivalent or at least overlapping. The binding of DEX to these high affinity sites may prove to be important, since the systemic administration of this steroid was found to down-regulate both type I and type II receptors in a number of brain regions. Because coadministration of the type I receptor antagonist RU26752 was shown to block these actions on type I, but not type II receptors, the formation of the DEX-type I receptor complex appears to be required for DEX-induced type I receptor down-regulation. An analysis of the in vitro efficacy of ALDO- vs. DEX-type I receptor transformation suggests that whereas there is a significant increase in the binding of both complexes to DNA-cellulose after treatment with thiocyanate, there is also a dramatic decrease in the stability of DEX- but not ALDO-type I receptor binding. We contend that it is this decrease in binding stability that mediates the DEX-induced down-regulation of type I receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Novel erythromycin derivatives with aryl groups tethered to the C-6 position are potent protein synthesis inhibitors and active against multidrug-resistant respiratory pathogens.

A novel series of erythromycin derivatives has been discovered with potent activity against key respiratory pathogens, including those resistant to erythromycin. These compounds are characterized by having an aryl group tethered to the C-6 position of the erythronolide skeleton. Extensive structural modification of the C-6 moiety led to the discovery of several promising compounds with potent activity against both mef- and erm-mediated resistant Streptoccoccus pneumoniae. Preliminary mechanistic studies indicated that the new macrolides are potent protein synthesis inhibitors, which interact with methylated ribosomes isolated from resistant organisms. In experimental animal models, these compounds exhibited excellent in vivo efficacy and balanced pharmacokinetic profiles.

Measles pneumonia. Treatment of a near-fatal case with corticosteroids and vitamin A.

A 29-year-old woman experienced overwhelming rubeola pneumonia requiring endotracheal intubation and mechanical ventilation. Treatment with high-dose corticosteroids and vitamin A was accompanied by a prompt clinical response. Further investigation of this novel therapy is needed.

Central venous catheter-related Corynebacterium minutissimum bacteremia.

Although Corynebacterium minutissimum is well-known as the cause of erythrasma, it is noted as the etiologic agent of nondermatologic disease only rarely. We document this organism as a cause of central venous catheter-associated bacteremia and report the use of pulsed-field gel electrophoresis to characterize its molecular epidemiology.

Environmental contamination with vancomycin-resistant enterococci in an outpatient setting.

Eleven cancer patients colonized with vancomycin-resistant enterococci (VRE) were followed as outpatients. Environmental cultures were obtained from clinic rooms before and after patients care. Environmental contamination occurred in 29% of encounters. Superficial disinfection did not eradicate contamination, although more thorough cleaning did. We conclude that environmental VRE contamination occurs in the outpatient setting. Infection control practices, similar to those used in the inpatient setting, may be necessary for outpatient clinics.

Outbreak of vancomycin-resistant Enterococcus faecium in a neonatal intensive care unit.

An outbreak of vancomycin-resistant Enterococcus faecium involving 28 infants in a neonatal intensive care unit was observed. Successful control of the outbreak was achieved following use of patient and staff cohorting, contact isolation precautions, patient and environmental surveillance cultures, environmental decontamination, molecular typing, introduction of an alcohol-based hand disinfectant, and decreased use of vancomycin.

Synthesis of 2-fluoro-6-O-propargyl-11,12-carbamate ketolides. A novel class of antibiotics.

A novel class of 2-fluoro-6-O-propargyl-11,12-carbamate ketolide derivatives of erythromycin has been synthesized for antibacterial SAR studies. Replacement of the C2-hydrogen by a fluorine atom allows the synthesis of 6-O-propargylic ketones and electron-deficient 6-O-propargylic aromatic derivatives by preventing intramolecular C2-enolate Michael cyclization.

Immortalized B-lymphocytes from rheumatoid synovial tissue show specificity for bacterial HSP 60.

Several studies indicate a pathogenetic role of T-lymphocytes with specificity for heat shock proteins (HSP) in rheumatoid arthritis (RA). Surprisingly, there are no experimental data for B-lymphocytes with specificity for HSP. To investigate whether B-lymphocytes from rheumatoid synovial tissue show a specificity for HSP 60 we immortalized synovial tissue B-lymphocytes by the electrofusion technique and tested the specificity of the B-cell clones for HSP 60 by ELISA. Tissue samples from four patients with classic, active RA were used in this study. The isolated cells were electrofused in strongly hypo-osmolar medium with cells either of the mouse strain X63-Ag8-653 (Ag8) or the heteromyeloma strain HAB-1. Clones positive for IgG, the IgG fraction of the supernatant of the isolated synovial cells and the IgG of the serum of the patients were tested in an ELISA for reactivity to the recombinant HSP 60 or Yersinia enterocolitica, which shows great homology with mycobacterial HSP 65 and human HSP 60. The expression of this HSP 60 was studied in normal and rheumatoid synovial tissue using a polyclonal rabbit serum against HSP 60 from Y. enterocolitica (Ye HSP 60). In this way we investigate differences in the expression of HSP 60 and compared the pattern of this HSP60 with the pattern of mycobacterial HSP65 and human HSP 60 described by others. In three of four patients 10 IgG secreting B-cell clones showing a specificity for HSP 60 were detected. IgG specific for HSP 60 was also detected in the supernatant of the isolated synovial cells before fusion and in the serum of these patients. HSP 60 was demonstrated immunohistochemically within the rheumatoid synovial tissue and showed stronger expression with a different distribution when compared with the expression in normal synovial tissue. B-cell clones from rheumatoid synovial tissue thus exhibit a specificity for bacterial HSP 60, and a monospecific rabbit serum against this HSP shows strong reactivity within the rheumatoid synovial tissue. It may be postulated that a humoral HSP 60 response, initially directed against an infectious agent, could react with cross-reactive epitopes of rheumatoid synovial tissue or with self-HSP perpetuating the local inflammatory process.

Ischemia induces a selective biphasic response in brain mitochondrial mRNA levels.

We determined the independent effects of hypoxia, glucose deprivation and ischemia (hypoxia plus glucose deprivation) on steady-state levels of mRNA coding for specific nuclear and mitochondrially encoded enzymes of oxidative metabolism in cultured rat neurons and glia. Neither hypoxia nor low glucose alone changed steady-state message levels for any transcript. However, ischemia induced a biphasic effect on mitochondrially encoded transcripts for cytochrome oxidase subunit two (CO2) and the subunits 8 and 6 of ATPase (A 8/6), initially decreasing and then increasing mRNA levels to or above the levels recorded prior to ischemia. In contrast, three nuclear encoded transcripts for mitochondrial proteins were decreased by ischemia. These data demonstrate a lack of coordination between the expression of nuclear and mitochondrial genes in the initial response to ischemia and suggest that a selective, primary reaction to brain cell insults exists within the mitochondrion.

Hyperbaric oxygenation increases arousal and breathing movements in fetal lambs.

Oxygenation produced by distending the lungs with 100% O2 increases the occurrence of arousal and fetal breathing movements (FBM), particularly during non-rapid-eye-movement (NREM) sleep, in fetal sheep of > or = 135 days of gestation. We studied the breathing and behavioral responses to a rise in arterial PO2 (PaO2) without lung distension in fetuses between 128 and 132 days of gestation. Twelve fetuses were chronically instrumented to record FBM, behavioral state, blood pressure, arterial blood gas tensions, and pH. Fetal PaO2 was raised by having the ewe breathe 100% O2 at 3 atmosphere absolute pressure spontaneously (group 1, n = 5, 129 +/- 1 days of gestation) or with mechanical ventilation to control fetal arterial PCO2 (group 2, n = 7, 131 +/- 1 days of gestation). Hyperbaric oxygenation raised fetal PaO2 by 20 Torr in both groups. During hyperbaric oxygenation, the occurrence of arousal increased severalfold in both groups. The occurrence of FBM increased during arousal in both groups, during rapid-eye-movement sleep in group 1, and during NREM sleep in group 2. The timing of diaphragmatic activity during arousal and the variability of diaphragmatic activity during NREM sleep were different than those in rapid-eye-movement sleep. We conclude that oxygenation without lung distension increases the occurrence of arousal and of FBM, principally during arousal and NREM sleep, in fetuses of < or = 135 days of gestation.