Investigations of cellular processes demand immediate arresting of the process at any given time and excellent retention of cellular material and excellent visibility of membranes. To achieve this goal we used cryofixation to arrest cellular processes instantly and tested diverse freeze-substitution protocols. Madin-Darby kidney cells and Vero cells were grown on carbon-coated sapphire disks. For cryofixation the sapphire disks covered with a cell monolayer were injected with the aid of a guillotine into liquid propane or ethane or a mixture of both cooled by liquid nitrogen. Freezing of the cryogen was prevented by using a partially insulated cylinder and by vigorous stirring that results in a substantial decrement of the freezing point of the cryogen. Cell monolayers can be cryofixed successfully using the guillotine in a safety hood at ambient temperature and humidity or at 37 degrees C and 45% humidity. The freezing unit can also be placed in a laminar flow for working under biohazard conditions. For visualizing cell membranes at high contrast and high resolution, cells were substituted in the presence of various concentrations of glutaraldehyde and osmium tetroxide and the temperature was raised to diverse final temperatures. Substitution for 4 hours at -90 degrees C in anhydrous acetone containing 0.25% anhydrous glutaraldehyde and 0.5% osmium tetroxide followed by a temperature rise of 5 degrees C/hour to 0 degrees C and final incubation for 1 hour at 0 degrees C resulted in high contrast and excellent visibility of subcellular components at the level of the membrane bilayer. The high spatial and temporal resolution makes this methodology an excellent tool for studying cell membrane-bound processes, such as virus-cell interactions.

Background: The epidemiology of methicillin-resistant

Small colony variants of Staphylococcus aureus can cause persistent and recurrent infections. There are only a few reports of small colony variants of coagulase-negative staphylococci. Herein a case of infection with a teicoplanin-resistant small colony variant of Staphylococcus epidermidis is presented. The small colony variant was isolated from blood cultures of a patient with acute leukaemia and therapy-induced neutropenia who was treated with vancomycin for catheter-associated bloodstream infection. Despite removal of the catheter and adequate antibiotic therapy, the infection did not clear and the patient died 20 days after continuous antibiotic therapy.

ObjectivesPlasmid-mediated AmpC beta-lactamase-producing (pAmpC) Enterobacteriaceae are increasing worldwide, difficult to identify and often confounded with extended-spectrum beta-lactamase (ESBL) producers. The low prevalence precludes routine universal admission screening. Therefore, we evaluated potential risk factors for carriage of pAmpC-producing Enterobacteriaceae that would allow targeted screening to improve yield and reduce cost.Patients and methodsWe performed a case control study at a tertiary care center from 1/2006 to 12/2010. Cases were adult patients in whom pAmpC-producing Enterobacteriaceae were isolated; controls were chosen among carriers of ESBL-producing Enterobacteriaceae. Both infected and colonized patients were included.ResultsOver five years, we identified 40 pAmpC producers in 39 patients among 16,247 screened consecutive isolates of Enterobacteriaceae. The pAmpC prevalence was low (0.25%), but more than 30% of pAmpC carriers received incorrect empirical antibiotic treatment. When compared with 39 ESBL controls, pAmpC carriage was associated with clinically confirmed infections in 74% (versus 51%) (p=0.035), mainly of the urinary tract, previous antibiotic exposure in 63% (versus 36%) (p=0.035) and carriage of a nasogastric tube in 23% (versus 0%) (p=0.002). In the multivariate regression analysis only clinically confirmed infections remained significantly associated with pAmpC carriage (OR 1.44 (95%CI 1.15-2.57)). No other clinical and blood test-associated risk factor allowed discrimination of pAmpC-carrying patients from ESBL controls. The type of acquisition – nosocomial versus community-acquired – was also non-informative for resistance type, as 46% of pAmpC- and 44% of ESBL-producing Enterobacteriaceae were community-acquired.ConclusionsThis study could not identify a clinical profile that would allow targeted screening for pAmpC-producing Enterobacteriaceae when compared to ESBL carriers. Because empiric antimicrobial therapy was inappropriate in more than 30%, rapid identification of pAmpC carriers is needed. New microbiological methods are therefore required to simplify rapid and reliable detection of pAmpC carriers.

No abstract

In this rat model of localized invasive pulmonary aspergillosis effects of early resection on survival could be demonstrated only in animals not receiving amphotericin B treatment.

Rapid and sensitive detection of mycobacteria is essential for the successful treatment and control of mycobacterial infections. It is considered good laboratory practice to combine a liquid medium with at least one solid medium for culturing mycobacteria from clinical specimens. Automated systems with liquid media provide faster results and are more sensitive, while solid media help to detect mixed infections and can detect mycobacteria that do not grow in the broth medium. In order to evaluate the BacT/ALERT 3D system (bioMérieux, Durham, NC, USA) and to find the optimal combination of media, we compared the results achieved with BacT/ALERT 3D, Lowenstein-Jensen (L-J) medium (Bio-Rad, Marne-la Coquette, France), and the Middlebrook 7H10/7H11 biplate (Becton Dickinson, Sparks, MD, USA) on consecutive clinical specimens collected within a period of 26 months. A total of 6,250 clinical samples (4,051 respiratory specimens, 1,209 normally sterile body fluids, 292 biopsy specimens, 247 blood specimens, 344 urine specimens and 304 other specimens) from 3,247 patients were tested for the presence of mycobacteria. Specimens with a different panel of media (blood and urine) and specimens contaminated with bacteria other than mycobacteria were excluded from the evaluation. Tissue specimens were crushed and homogenized. Homogenized tissue, respiratory specimens and other specimens from contaminated sites were decontaminated with sodium dodecyl sulfate-NaOH as described by Tacquet and Tisson [1]. Liquids from sterile sites were concentrated by centrifugation (3000 g, 15 min). Aliquots of 0.3-0.5 ml were inoculated onto a L-J slant, a Middlebrook 7H10/7H11 selective agar biplate with carbenicillin, polymyxin B, trimethoprim lactate, and amphotericin B, and into a BacT/ALERT MP bottle. For specimens from

Among 18,424 blood culture sets processed during a study period of 18 months, 85 bottles that were positive by the BacT/ALERT system were Gram stain negative. Both acridine orange staining and subcultures detected microorganisms in a total of 12 bottles. Acridine orange staining can replace subcultures of false-positive blood cultures.Rapid detection of microorganisms in blood cultures is one of the most important tasks of the clinical microbiology laboratory. Modern automated blood culture systems have increased the isolation rate of microorganisms and shortened the time to detection. The BacT/ALERT system (BioMerieux Inc., Durham, N.C.) is based on the colorimetric detection of CO 2 produced by the growing microorganisms. The culture medium, detection system, and detection algorithm are designed to maximize the sensitivity of the system. A detection algorithm which maximizes sensitivity invariably leads to a certain percentage of false-positive results, i.e., cultures that are reported as positive by the system although no microorganisms can be detected by Gram staining or subcultures. Subculturing false-positive blood culture bottles is time-consuming and costly. Acridine orange (AO) staining has been described as a replacement for subculturing false-positive blood cultures with the BACTEC NR 660 system (1, 2). However, it is not possible to apply these findings to the BacT/ALERT system, since BacT/ALERT FA and FN bottles contain absorbent charcoal particles, which make the interpretation of Gram-stained and AO-stained smears difficult (7). In order to assess the need to perform routine subcultures of false-positive blood cultures with the BacT/ALERT system, we undertook a prospective study of all blood cultures that were reported as positive by the system but were Gram stain negative.BacT/ALERT FA aerobic and BacT/ALERT FN anaerobic blood culture bottles were incubated in the BacT/ALERT Classic instrument for 7 days. From each bottle reported positive by the system an aliquot of broth was Gram stained. Before the Gram staining the slides were heat fixed. AO stain (Difco Becton Dickinson, Sparks, Mass.) was performed on all BacT/ALERT-positive, Gram stain-negative blood culture bottles according to the instructions of the manufacturer. Gramstained and AO-stained slides were read by routine bench technicians. In addition, the bottles were subcultured to chocolate agar (incubated at 35°C in 5% CO 2 for 4 days), Sabouraud agar (incubated at 28°C under aerobic conditions for 4 days), and blood agar (incubated at 35°C under anaerobic conditions for 4 days).During an 18-month period (5 April 2001 to 4 October 2002), 18,424 sets of blood cultures were processed. Of these 2,074 (11.3%) were true positives. A total of 85 blood culture bottles were reported as positive by the system but were Gram stain negative. Upon subculturing, 73 of them showed no growth. Thus, the overall rate of false-positive blood culture bottles was 0.2% (0.3% for aerobic and 0.1% for anaerobic bottles). In previous studies, false-positive rat...