Pseudomonas aeruginosa and other gram-negative isolates from patients with cystic fibrosis (CF) may be difficult to identify because of their marked phenotypic diversity. We examined 200 gram-negative clinical isolates from CF respiratory tract specimens and compared identification by biochemical testing and real-time PCR with multiple different target sequences using a standardized combination of biochemical testing and molecular identification, including 16S rRNA partial sequencing and gyrB PCR and sequencing as a "gold standard." Of 50 isolates easily identified phenotypically as P. aeruginosa, all were positive with PCR primers for gyrB or oprI, 98% were positive with exotoxin A primers, and 90% were positive with algD primers. Of 50 P. aeruginosa isolates that could be identified by basic biochemical testing, 100% were positive by real-time PCR with gyrB or oprI primers, 96% were positive with exotoxin A primers, and 92% were positive with algD primers. For isolates requiring more-extensive biochemical evaluation, 13 isolates were identified as P. aeruginosa; all 13 were positive with gyrB primers, 12 of 13 were positive with oprI primers, 11 of 13 were positive with exotoxin A primers, and 10 of 13 were positive with algD primers. A single false-positive P. aeruginosa result was seen with oprI primers. The best-performing commercial biochemical testing was in exact agreement with molecular identification only 60% of the time for this most difficult group. Real-time PCR had costs similar to those of commercial biochemical testing but a much shorter turnaround time. Given the diversity of these CF isolates, real-time PCR with a combination of two target sequences appears to be the optimum choice for identification of atypical P. aeruginosa and for non-P. aeruginosa gram-negative isolates.Pseudomonas aeruginosa is the most significant pathogen affecting patients with cystic fibrosis (CF). It is often acquired in the first few years of life and causes chronic airway infections, which ultimately result in death. While P. aeruginosa is not thought of as an organism that is difficult to identify in the microbiology laboratory, CF isolates often have unique phenotypes, including the loss of pigment production, synthesis of rough lipopolysaccharide devoid of O side chains, and development of mucoidy (18). While the presence of mucoidy may be helpful in identifying CF isolates of P. aeruginosa, some of the other characteristics may make these isolates more difficult to identify. Both commercial methods of identification and standard biochemical testing may be lengthy and inaccurate (10, 11). In addition, CF patients often have other nonfermenting gram-negative bacilli isolated from their respiratory secretions, making rapid isolation and identification of P. aeruginosa even more difficult (1, 7). While sequencing 16S rRNA genes in CF isolates of P. aeruginosa and other gram-negative bacilli has utility (6), it may be expensive and time-consuming.The Cystic Fibrosis Foundation-funded Therapeutic Development Network (...

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A previously healthy 9-year-old girl presented with a 1-week history of left neck mass and low grade fever. Surgical drainage specimens from the mass were bacterial culture-negative. Legionella micdadei infection was diagnosed by a two step PCR-nucleic acid sequencing method and supported by organism-specific serology. A backyard hot tub was suspected as the source of infection.

A mouse model is described in which intraperitoneal injection of Fusobacterium necrophorum results in chronic liver abscesses. Viable bacterial counts from mouse lung, liver, and spleen were obtained after whole organ homogenization. From 2 h to 5 days postchallenge, liver was found to contain more bacteria than lung on a per gram basis. Bacterial counts from liver and spleen were about the same during the first 8 h; thereafter liver was found to contain more bacteria. By day 13, though bacterial counts were equivalent in the three organs, abscesses were only observed in liver. This predilection for the liver may be due to a nutritional and/or microenvironmental factor(s). Blood cultures of infected mice revealed a general lack of bacteremia. Extended immunization with formalin-killed cells was found to protect mice against F. necrophorum infection.