We report herein the detection of intracellular bacteria in phagocyte-smears obtained from septicemia-suspected blood samples by in situ hybridization . This was obtained by using nicktranslated biotin-11-dUTP-labeled DNA probes and streptavidin-alkaline phosphatase conjugates for visualization of the hybridized signals. The probes were made from random genomic DNA clones of bacteria which are frequently the causative agents of bacteremia, such as Staphylococcus spp ., Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Klebsiella spp . and Enterobacter spp. When our in situ hybridization method was compared with conventional culture protocols for the ability to detect bacteria from the blood of patients suspected of having septicemia , 30 positive results were obtained in 50 specimens by in situ hybridization methods. In contrast , only 7 positive results were obtained by blood cultures. Thus, even if bacteria cannot be detected by conventional blood cultures and histology, our in situ hybridization method allows for direct observation of bacterial foci in circulating phagocytes and identification of the bacteria . Our investigations suggest that in septicemia, circulating polymorphonuclear neutrophils carry some surviving bacteria as well as metabolized bacterial DNA and RNA for a considerable period of time. Thus , our in situ hybridization method using the phagocyte-smears have diagnostic value for detecting most bacteria which cause septicemia.
Prins cyclization of homoallylic alcohols, thiols, and amines with various aldehydes in an ionic liquid HF salt (Et(4)NF.5HF) afforded the corresponding 4-fluorinated heterocycles in excellent yields.
To differentiate Chlamydia spp., a primer pair designed to generate a genus-specific region of the major outer membrane protein (MOMP) gene was used in a PCR to amplify a single DNA fragment of 245-259 bp. In the PCR, the expected single DNA fragment was amplified from strains of Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, respectively. By restriction endonuclease analysis with Alul and PvuII, the amplified products exhibited four distinct patterns, corresponding to the four species. It is, therefore, concluded that one-step PCR followed by restriction endonuclease analysis as described in this study could be a valuable method for the detection and differentiation of Chlamydia species.
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