Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes targetingIdentification of Staphylococcus aureus in blood cultures begins with presumptive identification of gram-positive cocci in clusters (GPCC) in the Gram-stained smear of blood culture bottles that signal a positive result, whereas final identification must await subculture and overnight incubation (1,8). This delayed identification often results in empirical antibiotic therapy administered to patients with GPCC-positive blood cultures, although the majority of GPCC-positive blood cultures are later identified as coagulase-negative staphylococci (CoNS), such as Staphylococcus epidermidis, a common blood culture contaminant. Direct identification of S. aureus in GPCC-positive blood culture bottles may provide important diagnostic information, which would allow the selection of an appropriate course of treatment in a timely manner.Immunological, tube coagulase, and stable-endonuclease methods routinely used for identification of S. aureus following subculture have been applied directly to GPCC-positive blood culture bottles, but with variable sensitivities and specificities (6, 9, 10). In addition, molecular techniques, such as hybridization protection (3), fluorescence in situ hybridization (FISH) (5), and PCR (2) have been described for identification of S. aureus directly from positive blood cultures. In general, all studies have been performed on just a single blood culture medium and, therefore, do not address the potential for interference from different blood culture media, which in fact might explain the variable results. Furthermore, none of the studies involve blood culture media supplemented with charcoal, such as that used in the FAN BacT/Alert medium (bioMerieux, Hazelwood, Mo.), or resins, such as that in the BACTEC Plus medium (Becton Dickinson, Sparks, Md.). Those supplements may interfere with assays for direct identification of positive blood culture organisms.FISH with a peptide nucleic acid (PNA) probe to target 16S rRNA of S. aureus is a novel method for the rapid and specific identification of S. aureus directly from GPCC-positive blood cultures. S. aureus PNA FISH had a 97% sensitivity and 100% specificity compared to conventional methods when tested with the BacT/Alert blood culture system and FAN medium (7). The aim of the present study was to perform a blinded comparison of S. aureus PNA FISH with conventional methodology on blood cultures representing the ESP medium (Trek Diagnostic Systems, Inc., Westlake, Ohio), BACTEC medium (Becton Dickinson), and BacT/Alert medium (bioMerieux). Routine positive blood culture bottles in which gram-positive cocci in clusters were observed in Gram-stained smears were randomly collected at each of eight clinical microbiology laboratories. For the study, one smear for future testing was prepared from each blood culture shortly after the Gramstaining results were known. The smears are stable at room temperature for several weeks and were collected over a 1-to 2-week pe...

To determine the strain variation and persistence among isolates of methicillin-resistant Staphylococcus aureus (MRSA) cultured from patients with colonization over extended time spans, pulsed-field gel electrophoresis was used to analyze the isolates from 47 patients for whom at least two mecA-positive isolates collected a minimum of six months apart were available. For 22 (47%) patients, the isolates represented multiple distinct strains of Staphylococcus aureus, while 20 (43%) patients had only a single strain detected; five (11%) patients had similar, genetically related isolates. MRSA were frequently associated with mucocutaneous abnormalities; 29 (62%) patients had focal cutaneous defects, and ten (21%) had chronic dermatitis. Multiple strains of MRSA were detected more frequently than single strains among patients in whom the initial focus of MRSA resolved clinically and another mucocutaneous defect subsequently developed compared to patients with clinically persistent foci (11/15 versus 9/23, respectively; p = 0.05, Fisher's exact test). Among the 21 patients in this series for whom isolates cultured within a two-month time span were available, there were seven (33%) patients with multiple strains of MRSA, including one patient with polyclonal bacteremia. In summary, patients with long-term MRSA colonization often have several different strains of MRSA, which typically change overtime in association with removal or resolution of a colonized focus and the recurrence of mucocutaneous defects.

Klebsiellae are an important cause of nosocomial infections. The two clinically relevant species, Klebsiella pneumoniae and KlebsieUla oxytoca, are differentiated by the ability to produce indole from tryptophan, K. oxytoca being indole positive. We report here the detailed biochemical and molecular analysis of two isolates of KiebsieUla, cultured from the same urine specimen, that differed only in their ability to produce indole. The two isolates were identical as determined by ribotyping and pulsed-field gel electrophoresis, and they differed from 10 epidemiologically unrelated strains. Probing with the Escherichia coli tryptophanase operon, tna, revealed seven restriction fragment length polymorphisms (RFLP) among the 12 strains. The two index strains had identical RFLP; no single RFLP could account for all of the indole-positive or-negative strains. Thus, the identification of epidemiologically related strains ofKlebsieUla differing only in indole production may warrant further examination to determine whether the strains are clonal. Klebsiellae are an important cause of nosocomial infections and have been responsible for epidemic infections, particularly in intensive care units (6, 11). In this context, the accurate identification of organisms is critical in defining nosocomial spread among patients. Within the genus Klebsiella, Klebsiella pneumoniae and Klebsiella oxytoca account for the majority of clinical isolates, differing biochemically in that K oxytoca is able to produce indole from tryptophan. We describe here the analysis of two isolates of Klebsiella that differed phenotypically only in their ability to produce indole from tryptophan. The genetic relationship between the isolates was determined by pulsed-field gel electrophoresis (PFGE) and Southern blot analysis after probing with the Escherichia coli ribosomal operon (ribotype). In addition, polymorphisms of the tryptophanase locus were evaluated by probing with the E. coli tryptophanase operon (4).

The application of molecular typing clearly demonstrated clonality among the isolates and indicated that a common source of contamination, most likely the CMGB tubes, was responsible for these cases.