Hiroshi Ueyama

Detection of Bacteria in Phagocyte-Smears from Septicemia-Suspected Blood by In Situ Hybridization Using Biotinylated Probes

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 nick‐translated 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.

Detection of Staphylococci in Mouse Phagocytic Cells by in Situ Hybridization Using Biotinylated Dna Probes

In situ hybridization was used to detect intracellular Staphylococcus aureus and S. epidermidis in mouse phagocytic cells after experimental infection of C3H mice with Staphylococci via abdominal or intravenous injection. Isolated ascites or whole blood were tested by the phagocyte smear technique, using bacteriolytic enzymes to preserve phagocytic cell morphology. The exposed bacterial DNA was visualized as intracellular hybridized signals by use of biotinylated DNA probes and by immunocytochemistry using streptavidin-alkaline phosphatase conjugates as detector molecules. These DNA probes, prepared from randomly cloned genomic DNA fragments of S. aureus and S. epidermidis, were strain-specific and did not cross-hybridize either in situ or on dot-blot hybridization. This technique of in situ hybridization with phagocyte smears is useful for detection and diagnosis of intracellular bacteria regardless of viability.

Binding of Streptavidin to Bacteria or Fungi and Its Applications in Detecting These Microbes

We have investigated the characteristics and utilities of streptavidin‐binding to gram‐negative and gram‐positive bacteria and Candida spp. The pre‐treatment of these microbes with chemical reagents such as CHCl3, NaOH, and Tween 20 have allowed colorimetric visualization under light microscopy or quantitation on nitrocellulose membranes, using streptavidin/biotinylated alkaline phosphatase conjugates. Analysis of this binding was confirmed by western blot. These binding reactions were due to the specific interaction of streptavidin with biotinylated proteins present in the microbes. Competition assays with free biotin or inhibition by an antibiotin antibody confirmed binding to these proteins. With knowledge of these strongly specific interactions, we attempted to reveal the biotinylated proteins within these microbes using clinical specimens. Using phagocyte‐smears from blood, urine, and ascites, these intracellular microbes were easily detected by light microscopy. One of the septic blood samples stained by our technique revealed semi‐digested microbial signals despite the absence of a signal with routine staining. This detection system, which combines streptavidin as a probe and biotinylated proteins as a microbial marker, is useful in staining for intracellular bacteria or fungi (e.g., microbial infections in phagocyte‐smears).