Phylogenetic placement and characterization of a new alpha-2 proteobacterium isolated from a patient with sepsis

Abstract

An alpha-2 proteobacterium, previously unknown as determined by its phylogenetic characteristics and the DNA sequence of its 16S rRNA gene, was isolated from a patient who presented an unusual clinical picture, including high remitting fever and multiorgan involvement. The bacterium was detected in multiple plasma samples, obtained during the acute phase of the disease, after cocultivation in cell culture media. Electron microscopy of the organism showed a three-layer laminar cell wall and electron-dense granules within the cytoplasm, as well as a polar flagellum. By means of PCR followed by sequencing of amplified 16S ribosomal DNA fragments, the bacterium was found to differ from all species for which ribosomal sequence information is available. It is here provisionally named the Rasbo bacterium. At a subsequent relapse, the bacterium was identified in pericardial fluid both by PCR/sequencing and by direct electron microscopy. At a second relapse, it was again cultured from plasma. After in vitro adaptation to solid media, the MICs of various antibiotics could be determined. A transient immunoglobulin M (IgM) but no IgG response to the bacterium was found by an indirect immunofluorescence test, as well as by an immobilization test during the acute phase of the disease. MATERIALS AND METHODSCase report. A previously healthy 33-year-old man was admitted to the hospital 8 days after the onset of respiratory symptoms and severe myalgia followed by the development of fever. One of the most conspicuous clinical features was remitting fever continuing for 3 months and with extreme quotidian amplitude, with peak values reaching 42.3°C. Severe symptoms involving serous membranes and visceral organs gradually developed, i.e., profuse diarrhea and pericardial, pleural, and peritoneal effusions, as well as signs of myocardial, hepatic, and splenic involvement. Intensive care was required for several weeks. An extensive search for a microbial cause by conventional methods failed to demonstrate an infectious agent, and broad-range testing for systemic, endocrine, and malignant diseases resulted in negative findings. Presumptive treatment was given with a great number of antibacterial drugs, with no clear response to any specific drug. After 3 months, when antimicrobial chemotherapy had been discontinued due to suspected side effects, the fever and other symptoms gradually disappeared and the patient could leave the hospital. In the meantime, an infectious agent from the patient's plasma had adapted to in vitro growth on artificial media (see below). Two months after the patient was discharged from the hospital a relapse occurred, and the patient was readmitted with chest symptoms and a temperature above 40°C. Seven hundred milliliters of pericardial fluid was evacuated. The patient recovered within 2 weeks on doxycycline treatment that was continued until a second relapse occurred 7 months later. Compliance was checked by determination of the doxycycline concentration in serum on admission at the second relapse. At this time...

Bartonella vinsoniisubsp.berkhoffiiand Related Members of the Alpha Subdivision of theProteobacteriain Dogs with Cardiac Arrhythmias, Endocarditis, or Myocarditis

“…Specifically, low or undetectable levels of Bartonellaspecific antibodies have been observed in culture-positive animals or human patients, even when the homologous organism is used as the test antigen. A similar observation was made in the case of a Swedish patient infected with the Rasbo bacterium, who failed to develop a detectable IgG antibody response to the organism despite a chronic course of infection (5). Alternatively, failure to detect an IgG-specific immune response to these organisms may reflect differences in cultureor tissue culture-grown organisms compared to the antigenic properties of the organisms in vivo.…”

“…Unexpectedly, when the partial 16S rRNA gene sequence derived from blood or other tissue samples from the other seven dogs was compared to GenBank sequences for other, closely related bacteria, including B. henselae, B. vinsonii, Brucella canis, and the alpha proteobacterium provisionally designated Rasbo, all sequences clustered together and were most closely related to Brucella canis and Rasbo bacterium (Fig. 3) (5). Although several Brucella spp.…”

“…berkhoffii (Table 4). Amplicons derived from seven other dogs (cases 4 to 10) clustered among several alpha proteobacteria, including Rhizobium, Agrobacterium, Brucella, Methylobacterium, and the currently unnamed Rasbo agent (5). Given the lack of a bacterial isolate and the limited quantity of DNA available, it was not possible to definitively identify the bacteria in these seven dogs.…”

“…In 1993, our laboratory isolated from a dog with endocarditis a novel Bartonella subspecies (6) that was subsequently designated Bartonella vinsonii subspecies berkhoffii (American Type Culture Collection type strain 51672) (22). In 1997, a new alpha-2 proteobacterium, provisionally designated Rasbo bacterium, was isolated from a chronically febrile patient with pericardial effusion and clinical evidence of myocardial disease (5). Because these organisms are highly fastidious, molecular diagnosis by PCR amplification and direct sequencing, as reported in recent studies of human endocarditis (references 13 and 19 and this study), may be necessary to confirm alphaproteobacterial infection.…”

Isolation of new fastidious α Proteobacteria and Afipia felis from hospital water supplies by direct plating and amoebal co-culture procedures

“…The 16S rDNA sequence of the group IV strain 34619 di¡ered at 22 of 1380 sites (98.3% similarity) from that of the Rasbo bacterium (Table 5) [16]. In phenotypic terms, this strain di¡ered from the Rasbo bacterial strain in its positive catalase reaction and ability to reduce nitrates [16]. The 16S rDNA sequence of the group V strain 76712 di¡ered at 30 of 1380 sites (97.8% similarity) from that of A. caulinodans [17].…”