Severe systemic Bacillus anthracis infection in an intravenous drug user.

There has recently been an outbreak of injectional anthrax infection secondary to contaminated heroin use in the UK and Europe. We present a case of a 37-year-old man presenting with pain and swelling in the groin following injection of heroin into the area. He was initially treated for severe cellulitis, however, he failed to respond to appropriate antimicrobial therapy. He went onto develop a widespread rash; it was then that a diagnosis of injectional anthrax infection was considered. Appropriate investigations were initiated including serum sample and tissue biopsy, and the diagnosis was confirmed. Management included extensive surgical debridement and a prolonged course of combination antibiotic therapy. The authors summarise the important steps in diagnosis and the management options in patients presenting with this life-threatening infection.

Antifungal activity of fused Mannich ketones triggers an oxidative stress response and is Cap1-dependent in Candida albicans.

We investigated the antifungal activity of fused Mannich ketone (FMK) congeners and two of their aminoalcohol derivatives. In particular, FMKs with five-membered saturated rings were shown to have minimum inhibitory concentration (MIC90s) ranging from 0.8 to 6 µg/mL toward C. albicans and the closely related C. parapsilosis and C. krusei while having reduced efficacy toward C. glabrata and almost no efficacy against Aspergillus sp. Transcript profiling of C. albicans cells exposed for 30 or 60 min to 2-(morpholinomethyl)-1-indanone, a representative FMK with a five-membered saturated ring, revealed a transcriptional response typical of oxidative stress and similar to that of a C. albicans Cap1 transcriptional activator. Consistently, C. albicans lacking the CAP1 gene was hypersensitive to this FMK, while C. albicans strains overexpressing CAP1 had decreased sensitivity to 2-(morpholinomethyl)-1-indanone. Quantitative structure-activity relationship studies revealed a correlation of antifungal potency and the energy of the lowest unoccupied molecular orbital of FMKs and unsaturated Mannich ketones thereby implicating redox cycling-mediated oxidative stress as a mechanism of action. This conclusion was further supported by the loss of antifungal activity upon conversion of representative FMKs to aminoalcohols that were unable to participate in redox cycles.

Application of chip-based flow cytometry for amphotericin B and fluconazole susceptibility testing on Candida strains.

Chip-based flow cytometry is a rather new method that offers an easy, fast opportunity for examination of yeasts, such as Candida cells. In our study cell-chip technology was tested with ATCC Candida strains to determine their viability and susceptibility against antifungal agents, amphotericin B and fluconazole. We found this technology to be suitable for the detection of Candida cells, for the differentiation between dead and living cells, and for the determination of amphotericin B and fluconazole susceptibility of different Candida strains (Bouquet et al., Mycoses 55:e90-e96, 2012).

Salmonella sternoclavicular osteomyelitis in a patient with Crohn's disease.

We describe a 73-year-old man with Crohn's disease and previous sternotomies, who developed Salmonella sternoclavicular osteomyelitis subsequent to a Salmonella enteritidis sepsis and closed fracture of his clavicle. We include evidence from several cases related to sternoclavicular osteomyelitis, and Salmonella osteomyelitis. We continue by summarising the aetologies of these diseases, and risk factors that predispose to them.

Amphotericin B and fluconazole susceptibility of Candida species determined by cell-chip technology.

The aim of this study was to apply the microfluidic cell-chip technology for susceptibility testing. The cell-chip technology was tested with ATCC Candida strains to determine their viability and susceptibility against amphotericin B and fluconazole. Fungal cells were labelled by Sytox Green, and measurements were carried out in the cell chips of the Agilent Bioanalyzer 2100 system. Results obtained by the chip technology were compared with the standard macrodilution method and conventional flow cytometry. Determination of minimum inhibitory concentration values was based on the differentiation between living and dead cells. The cell-chip method was found to be suitable for the detection of Candida cells, for the differentiation between dead and living cells and for the determination of amphotericin B and fluconazole susceptibility of fungal cells. The minimum inhibitory concentration values obtained by the standard macrodilution, the flow cytometry and the cell-chip method showed good correlation.

Antifungal unsaturated cyclic Mannich ketones and amino alcohols: study of mechanism of action.

The antifungal activity of some known unsaturated Mannich ketones and their amino alcohols has been reported and the mechanism of antifungal action has been studied. The inhibition of the fungal ergosterol, chitin, protein synthesis and pseudohypha formation was investigated. According to our results, Mannich ketones can influence the development of pseudohyphae of Candida albicans strains. In addition, they are able to induce significant changes in the protein composition of this fungal strain. Some of our Mannich ketones have shown inhibitory effect on the fungal chitin synthase enzyme. QSAR studies were also carried out.

Characterisation of protein composition and detection of IgA in cervicovaginal fluid by microchip technology.

In this paper the application of microchip electrophoresis to examine the protein profile of cervicovaginal fluid and the detection of IgA heavy and light chains is presented. This method is a fast growing field of technology and ensures high-speed analysis requiring only microliters of sample. Proteins with wide range of molecular masses could be separated within 1 min. Cervicovaginal specimens of healthy women showed a complex protein pattern-containing several peaks in the 15-70 kDa region. sIgA is considered to be an important protein constituent of all mucosal surfaces. Detection of sIgA in cervicovaginal samples was achievable by microchip technology. Under reduced circumstances (induced by mercaptoethanol, a component of the denaturating solution) the disulfide bonds connecting IgA heavy and light chains are broken up and chains can be detected as separate peaks during electrophoresis. In 82.5% of the cases only the light chain of IgA could be detected in the clinical samples. The intact IgA heavy chain could be demonstrated in only 12.5% of the cases. Based on our data some conclusions were provided about the correlation of these patterns with the age of patients, pH of the cervicovaginal fluid, operations performed before sample collection and usage of oral contraceptives.

Investigation of the effects of PACAP on the composition of tear and endolymph proteins.

Pituitary adenylate cyclase activating polypeptide (PACAP) is widely distributed in ocular tissues, including the lacrimal gland. PACAP has been shown to influence the activity of several exocrine glands, but its effects on the composition of the tear film are not known yet. Similarly, the presence of PACAP has already been shown in the inner ear, but it is not known whether PACAP influences the composition of the endolymph. The aim of the present study was to investigate whether systemic injection of PACAP has any modulatory effects on the protein composition of the tear film and endolymph using chip electrophoresis and mass spectrometry analysis. Tear and endolymph samples were collected from rats and chickens, respectively, at various time points after systemic injection of PACAP. Fluid samples were further processed for chip electrophoretic studies. No difference was found in the protein composition of the endolymph between control and PACAP-treated animals. In contrast, tear samples showed a marked difference after PACAP treatment. Proteins in the molecular range 50-70 kDa, which showed a different chip electropherogram profile in every PACAP-treated sample, were further analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PACAP treatment induced a repression in certain keratins, while others were induced after PACAP injection. Furthermore, PACAP treatment decreased aldehyde dehydrogenase expression. The present study provides a base for further studies on the in vivo effects of PACAP on the composition of tear film. These investigations may have important clinical relevance because of the noninvasive sample collection, the correlation between tear proteins and ocular diseases, and the possible presence of biomarkers for both ophthalmological and systemic pathological conditions.

Microfluidic chip analysis of outer membrane proteins responsible for serological cross-reaction between three Gram-negative bacteria: Proteus morganii O34, Escherichia coli O111 and Salmonella Adelaide O35.

Bacterial strains have complex and individual antigenic structure, which provides basis for their serological identification. However, serological cross-reaction may occur when antibodies against a certain strain recognize other strains too. The molecular basis of this phenomenon is the expression of similar or identical antigenic epitopes on the surface of different bacterial cells. Such cross-reactions might harden the serological diagnosis of pathogenic bacteria. But it can be also advantageous, when antigens of non-pathogenic strains can be used in the serological examinations. Serological cross-reaction between three taxonomically different strains--Proteus morganii O34 (8662/64), Escherichia coli O111 and Salmonella Adelaide O35--have been described. It has been proven that it is based partially on the similar lipopolysaccharide structures of these pathogens. In this study the involvement of the outer membrane proteins of these strains in the serological cross-reaction is presented. Microfluidic chip technology was applied for the detection of common proteins, which provided fast and quantitative data about the proteins that might be responsible for serological cross-reaction. Two outer membrane proteins with apparent molecular mass of 36 and 41 kDa, respectively, could be detected in the profile of each strain, while individual dominating protein peaks have also appeared in the protein profiles. The presence of common protein antigens was proven by Western blotting.

Bacterial outer membrane protein analysis by electrophoresis and microchip technology.

Outer membrane proteins are indispensable components of bacterial cells and participate in several relevant functions of the microorganisms. Changes in the outer membrane protein composition might alter antibiotic sensitivity and pathogenicity. Furthermore, the effects of various factors on outer membrane protein expression, such as antibiotic treatment, mutation, changes in the environment, lipopolysaccharide modification and biofilm formation, have been analyzed. Traditionally, the outer membrane protein profile determination was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Converting this technique to capillary electrophoresis format resulted in faster separation, lower sample consumption and automation. Coupling capillary electrophoresis with mass spectrometry enabled the fast identification of bacterial proteins, while immediate quantitative analysis permitted the determination of up- and downregulation of certain outer membrane proteins. Adapting capillary electrophoresis to microchip format ensured a further ten- to 100-fold decrease in separation time. Application of different separation techniques combined with various sensitive detector systems has ensured further opportunities in the field of high-throughput bacterial protein analysis. This review provides an overview using selected examples of outer membrane proteins and the development and application of the electrophoretic and microchip technologies for the analysis of these proteins.

CE to monitor endotoxins by protein complexation.

A new CE method for fast and efficient analysis of bacterial endotoxins (lipopolysaccharides) is described. It is based on the strong interaction between proteins and endotoxins. The UV absorption of the protein component in the complex is used for the detection. The electrophoretic mobility of the complex hemoglobin/endotoxin can be employed for qualitative analysis of the endotoxin. For instance, the structural differences between "smooth" and "rough" lipopolysaccharides from Salmonella minnesota (wild-type), Salmonella minnesota R595 and Shigella sonnei R562H are reflected in the electrophoretic mobilities of their hemoglobin complex.

Molecular epidemiology of VIM-4 metallo-beta-lactamase-producing Pseudomonas sp. isolates in Hungary.

VIM metallo-beta-lactamase-producing serotype O11 or O12 Pseudomonas aeruginosa isolates infecting or colonizing 19 patients from seven hospitals in Hungary were characterized between October 2003 and November 2005. Macrorestriction analysis revealed the involvement of hospitals from three different towns in northwest Hungary in an outbreak caused by VIM-4-producing P. aeruginosa.

Effect of antifungal agents on protein composition of Candida albicans studied by capillary electrophoresis and chip technology.

In the present study protein profile of a Candida albicans strain had been examined by chip technology and conventional capillary electrophoresis (CE). Profiles could be characterised by the presence of ten dominating protein peaks. These proteins could be distinguished by both techniques, but their quantity showed significant differences in the electropherograms obtained by CE and chip method. Changes in the protein profile were induced by administration of different antifungal agents. Fluconazole and amphotericin B treatment was able to induce similar changes in the pattern, appearance of a 40-kDa protein and up-regulation of a 60-kDa protein was observed by chip technology. Increase in the quantity of these proteins under stress effect (antifungal treatment) might refer to their stress function in the fungal cell. Treatment of C. albicans cells with MK 94 (fused cyclic Mannich ketone) antifungal compound induced not only the previously mentioned changes, but further specific alterations, appearance of a 19-kDa protein and up-regulation of the low molecular weight proteins. This might refer to the different mode of action of this agent on the fungal cells. Conventional capillary electrophoresis was suitable to detect the appearance of the 19-kDa peak, and up-regulation of the 60 kDa protein, but the other changes could not be detected by this technique. Shorter running time, more effective and baseline separation of proteins refer to the advantages of microchip-based method in the analysis of complex biological samples.

In vitro study of antibiotic effect on bacterial adherence to acrylic intraocular lenses.

Implantation of artificial intraocular lenses into the eye during ophthalmic surgical procedures ensures an unliving surface on which bacterial pathogens may attach and form biofilms. Despite antibiotic treatment bacteria growing in biofilms might cause inflammation and serious complications. In this study the adhesive ability of 7 Staphylococcus aureus and 11 coagulase-negative Staphylococcus (CNS) strains to the surface of acrylic intraocular lenses had been examined by the ultrasonic method. In untreated cases adhesion of the S. aureus and CNS strains did not differ significantly. We could not demonstrate significant differences between the adhesive ability of the standard strains and the clinical isolates. In this study a single--60 min long--antibiotic (ciprofloxacin and tobramycin) treatment had been applied, that correlate well with the single or intermittant antibiotic prophylaxis of patients. Ciprofloxacin administration was able to reduce significantly the number of attached cells on the surface of acrylic lenses both in the case of S. aureus and CNS strains. Dependence of the effect from concentration could also be demonstrated. Tobramycin treatment was able to inhibit significantly the attachment of S. aureus cells. Despite the debate on antibiotic prophylaxis we presented in our experiments that a single antibiotic administration can decrease the attachment of bacterial cells to the surface of acrylic intraocular lenses, and might be effective in the prevention of postoperative endophthalmitis, that is a rare but serious complication of ophthalmic surgery.

Effect of iron restriction on outer membrane protein composition of Pseudomonas strains studied by conventional and microchip electrophoresis.

In this study the outer membrane protein (OMP) composition of six Pseudomonas aeruginosa strains had been analyzed by conventional CE and microchip electrophoresis. Bacterial OMPs are important virulence factors and play a significant role in the pathogenesis of infectious diseases. Changes in their composition might refer to the altered pathogenic properties and antibiotic sensitivity of a certain strain. Pathogenic bacteria invading the human host have to multiplicate under iron-restricted conditions that induce changes in the OMP composition. High-molecular-weight OMPs have to be expressed, which serve as receptors for the iron-siderophore complexes. OMP patterns of bacteria obtained by the two different methods in this study were similar, all major proteins could be detected by both techniques, and the molecular weights showed good correlations, although direct comparison of the peak areas is not straightforward due to the different detection methods (UV and LIF). Changes in OMP composition under iron restriction could be detected, and appearance of a 92 kDa protein in all six P. aeruginosa strains and a 94 kDa protein in the KT 2 strain could be demonstrated. Besides that up- and downregulation of certain proteins could be also detected. The increased separation speed, picoliters of sample consumption, baseline separation achieved more frequently by this method--especially in the high-molecular-weight region--showed the advantages of microchip electrophoresis in the analysis of clinical samples.

Effect of antibiotic treatment on bacterial attachment to a DePuy Enduron orthopedic implant.

BACKGROUND: The increasing incidence of bacterial infections in orthopedic surgery might be related to the increasing application of artificial devices. In most cases, bacteria multiply on the surface of implants in biofilms. Poor penetration of antibiotics, frequent necessity of prosthesis removal, chronic processes and financial costs emphasize the significance of preventive measures. METHOD: Adhesion of bacterial strains (two Staphylococcus aureus, two coagulase-negative staphylococci and two Pseudomonas aeruginosa strains isolated from orthopedic patients' wounds) to the surface of a polyethylene cup was investigated using an ultrasonic method. Results were compared to the adhesive ability of three Hungarian standard strains. The effect of antibiotic treatment (cefuroxime, cefotaxime, amoxicillin with clavulanic acid and amikacin) has been examined. RESULTS: The staphylococcal strains showed significantly higher adhesive ability than Pseudomonas strains. Antibiotic treatment significantly reduced the attachment of bacteria. The higher the concentration of the antibiotics, the higher was the decrease in bacterial adhesion. CONCLUSIONS: Antibiotic prophylaxis was proven to be effective against bacterial adhesion, and, if applied at the proper time at the highest tolerable dose, it might prevent the formation of biofilms.

Effect of antibiotics on cell surface hydrophobicity of bacteria causing orthopedic wound infections.

BACKGROUND: Despite antibiotic prophylaxis and treatment, the incidence of wound infections in orthopedic surgery is significant. Postoperative wound infection is a multifactorial process, which can be modified by several bacterial factors. Cell surface hydrophobicity of bacteria is a very important physicochemical feature, which has a great influence on the ability of bacteria to adhere to the surface of host cells or medical implants. METHODS: In this study, the hydrophobic properties of thirteen bacterial strains (coagulase-negative staphylococci, Staphylococcus aureus and Pseudomonas aeruginosa) isolated from patients with postoperative deep wound infections following orthopedic procedures were determined by the salt aggregation test. Results were compared to the hydrophobicity of three Hungarian standard bacterial strains. The modifying effect of four antibiotics (cefuroxime, cefotaxime, amoxicillin combined with clavulanic acid and amikacin)--applied most often in our Department for prophylaxis and treatment of patients--were analyzed. RESULTS: The cell surface hydrophobicity of certain strains showed considerable changes after antibiotic treatment. These alterations indicated the decrease in hydrophobicity. Supra-inhibitory concentrations (2x minimum inhibitory concentrations, MIC) of the antibiotics were able to induce more frequent alterations in hydrophobicity than sub-inhibitory (0.5x MIC) levels. CONCLUSIONS: Alterations in cell surface hydrophobicity caused by antibiotics can modify the adhesion process and thus the pathogenicity of bacterial strains. These changes should be taken into consideration in the management of proper antibiotic prophylaxis and in the treatment of orthopedic patients.

Capillary electrophoresis study of outer membrane proteins of Pseudomonas strains upon antibiotic treatment.

Nosocomial wound infections by Pseudomonas aeruginosa strains have increasing importance in orthopaedic surgery. Outer membrane protein composition and cell-surface hydrophobicity of the bacteria have strong influence on adhesion to living tissues or artificial medical devices. Outer membrane proteins of five Pseudomonas strains (KT 2, KT 7, KT 25, KT 28, KT 39) isolated from orthopaedic patients' wounds and one standard strain NIH Hungary 170000 isolated from pus were examined. The capillary electrophoretic patterns of the outer membrane proteins were characteristic to each bacterial strains possessing different relative ratios of major and minor proteins. Antibiotic treatment of bacteria with three antibiotics, cefotaximum, amoxicillinum/clavulinic acid and amikacinum (applied often in prophylaxis and treatment of patients) induced changes in the electrophoretic profiles showing that outer membrane protein composition was altered significantly. The most pronounced alterations in the electrophoretic patterns after antibiotic treatment were obtained in the cases of the strains KT 2, KT 7 and KT 28. The amikacinum administration strongly decreased the relative percentage of the 38800 rel. mol. mass protein in KT 2 (from 20 to 6%). while the relative amount of the same protein increased significantly in KT 7 and KT 28 after cefotaximum treatment (from 2 to 16% and from 12 to 28%, respectively). Decrease in cell-surface hydrophobicity was also observed by salt aggregation test. The results obtained can be used to determine the therapeutic concentrations of antibiotics to induce changes in the adhesion properties of bacteria.