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A Gram-negative rod was isolated from the blood cultures of an 84-year-old man with foot cellulitis. The bacterium was first identified as Sphingobacterium spiritivorum on the basis of standard assimilation tests. However, sequencing analysis of its 16S rRNA genes and whole genome hybridization studies with other related bacteria showed that this isolate belongs to a so far undescribed species of Sphingobacterium, close to S. mizutae. This bacterium was susceptible to most of the antibiotics tested, including glycopeptides, but was resistant to aminoglycosides and polymyxins. Treatment with amoxicillin–clavulanate cured the infection.
The genus Sphingobacterium consists of Gram-negative environmental bacilli that are rarely involved in human infections. Two species, Sphingobacterium multivorum and Sphingobacterium spiritivorum (previously classified as Flavobacterium spp.), have occasionally been associated with bacteremia [1–3], peritonitis  and chronic respiratory infections  in patients with severe underlying conditions. Here, we report the case of a patient without any evident risk factor at the time of admission who experienced a bacteremic episode caused by a so far undescribed Sphingobacterium species. The difficulties met in the identification of this opportunistic pathogen at the laboratory prompted us to report this case.
An 84-year-old man was admitted to the hospital because of a rash on his right leg, and a fever of 39.7 °C. On the day before admission, the patient had noticed the onset of erythematous plaques with edema, producing a burning sensation. Physical examination revealed enlarged and tender right inguinal lymph nodes. Blood analysis showed a high white blood cell count (35 × 103/mm3), with 91% neutrophils, decreased hematocrit of 30%, and increased mean corpuscular volume (109 F1). Serum γ-glutamyl transferase was found to be slightly elevated, the serum iron concentration was abnormally low (2 µmol/L), while serum vitamin B12 and folate status were normal. Three blood cultures were performed in order to document the cause of the sepsis, and the patient was treated intravenously with amoxicillin–clavulanate (1 g every 8 h) and paracetamol (2 g every 8 h). His fever resolved completely within 12 h. Gastroscopy revealed varices in the area of the cardia, which were probably implicated in the iron deficiency, while microscopic examination of biopsy specimens from the fundus confirmed gastric atrophy. The bone marrow smear suggested a refractory anemia.
After 3 days of incubation, the aerobic blood cultures (Castaneda; Unipath, Dardilly, France) yielded a non-motile, oxidase-positive, non-fermenting Gram-negative rod. The bacterium produced small, pale yellow colonies on Mueller–Hinton medium within 24 h at 37 °C, but was unable to grow at 42 °C or on MacConkey agar at 37 °C. Assimilation tests (API 20NE strip panels, bioMérieux, Marcy l'Etoile, France) identified this isolate as S. spiritivorum with 98.8% certainty. However, the API database contains only two Sphingobacterium species, namely S. spiritivorum and S. multivorum, which usually grow on MacConkey medium . Antimicrobial susceptibility testing was performed with the microdilution broth method (NEG MIC Microscan, Dade, Behring Inc., Sacremento, CA), as the disk method is known to provide unreliable data with flavobacteria [6,7]. Minimal inhibitory concentrations (MICs) of glycopeptides were determined by the Etest (AB-Biodisk Solna, Sweden) . The strain was found to be more susceptible than Chryseobacterium and Myroides species (former members of the genus Flavobacterium) to many drugs, including amoxicillin–clavulanate (MIC ≤ 2/1 mg/L), ticarcillin (MIC ≤ 8 mg/L), piperacillin (MIC ≤ 4 mg/L), cefotaxime (MIC ≤ 4 mg/L), ceftazidime (MIC = 2 mg/L), imipenem (MIC ≤ 0.5 mg/L), nalidixic acid (MIC ≤ 8 mg/L), norfloxacin (MIC ≤ 1 mg/L), ciprofloxacin (MIC ≤ 0.25 mg/L), trimethoprim–sulfamethoxazole (MIC ≤ 2/38 mg/L), vancomycin (MIC = 2 mg/L), and teicoplanin (MIC = 4 mg/L). It showed moderate susceptibility to amoxicillin (MIC = 4 mg/L), cephalothin (MIC = 32 mg/L), cefoxitin (MIC = 32 mg/L), and aztreonam (MIC = 32 mg/L). Finally, like other flavobacteria-like organisms, it was highly resistant to aminoglycosides and polymyxins (data not shown).
After 10 days of amoxicillin–clavulanate given intravenously at a dose of 1 g three times daily, the patient was considered to be cured of his infection, and was discharged with treatment for iron deficiency.
Although the genus Sphingobacterium is not included in the family Flavobacteriaceae, members of this genus are phenotypically similar to many of the flavobacteria and flavobacteria-like organisms. Sphingobacterium spp. are yellow-pigmented, oxidase-positive, indole-negative, saccharolytic, Gram-negative rods . Eight named species and two unnamed genospecies have been distinguished so far by genetic methods: S. multivorum (formerly F. multivorum, CDC group IIk-2), S. spiritivorum (includes species formerly designated as F. spiritivorum, F. yabuuchiae, and CDC group IIk-3), S. mizutae, S. thalpophilum, S. heparinum, S. faecium, S. antarcticus, S. piscium, and the unnamed species Sphingobacterium genospecies 1 and 2 [9–12]. Since our isolate appeared to be very different from S. heparinum, S. faecium, S. antarcticus, S. piscium and Sphingobacterium genospecies 1 and 2 according to assimilation tests (API 20NE) , a more complete characterization of the strain was carried out in a reference laboratory (Pasteur Institute, Paris). Its identification required both partial sequencing of 16S rRNA genes, and quantitative DNA–DNA hybridizations with type strains of the closest Sphingobacterium species. The 16S rRNA sequences deposited in GenBank of S. mizutae, F. yabuuchiae, S. spiritivorum, S. multivorum and S. thalpophilum diverge by 1.5–9.9%. Divergence between the 16S rRNA gene sequence of the isolate and that of related organisms was 4.3% with S. mizutae, 9.6% with F. yabuuchiae and S. spiritivorum, 10.2% with S. thalpophilum, 10.9% with S. multivorum, and 15.5% with F. canadiensis. These data indicated that the isolate belonged to the Sphingobacterium phylogenetic branch. DNA–DNA hybridization experiments using the chromosome of the isolate as the labeled DNA further demonstrated relatedness of 23% with S. mizutae, 13% with S. spiritivorum, and 11% with F. yabuuchiae. The relatedness with S. mizutae was found to be 18% when the chromosome of this latter species was used as the labeled DNA. Thus, these results provided evidence that the isolate belongs to a new species of the genus Sphingobacterium, close to S. mizutae.
The source of infection in the patient was probably environmental, although investigation failed to detect the bacterium in the skin lesions. The role of anemia in the development of the sepsis still remains unclear, but may have contributed to impairment of the host's immune defenses. In conclusion, the causative agent of this bacteremic episode was a so far undescribed Sphingobacterium species, capable of behaving as an opportunistic pathogen and producing severe infection in compromised patients. Identification of this organism at the species level required extensive molecular methods.