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Department of Clinical Microbiology, Karolinska University Hospital—Solna, SE-171 76 Stockholm, Sweden
ABSTRACT
We report on the isolation of two species of Actinobaculum from blood culture of a patient with chronic renal failure. The two isolates were distinct with regard to their morphological and biochemical characteristics. Subsequent 16S rRNA gene sequencing classified the two species as Actinobaculum schaalii and A. urinale.
CASE REPORT
A 68-year-old man with a history of chronic renal failure, pacemaker, atrial fibrillation, heart failure, hiatus hernia, and iron deficiency anemia was admitted to hospital because of an abrupt onset of frequent watery diarrhea, dehydration, fever, and oliguria progressing to anuria for the previous 5 days. On admission, the hemoglobin level was 104 g/liter (normal range, 130 to 160 g/liter), the serum creatinine level was 450 μmol/liter (normal range, 65 to 110 μmol/liter), the serum sodium level was 128 mmol/liter (normal range, 139 to 147 mmol/liter), the serum potassium level was 4.9 mmol/liter (normal range, 3.6 to 4.6 mmol/liter), and the C-reactive protein level was 284 mg/liter (normal range, <10 mg/liter). A computed tomography scan of the abdomen showed an obscure density at the region of the appendix. Blood, urine, and stool specimens for culture, as well as stool specimens for Clostridium difficile toxin detection, were submitted to the clinical bacteriology laboratory at the Karolinska University Hospital, Stockholm, Sweden. The patient was treated with a combination of metronidazole and piperacillin-tazobactam as well as intravenous fluid replacement and general supportive measures for management of the renal insufficiency. Diuresis was gradually restored, and the acute diarrheal symptoms as well as the fever subsided. The s-creatinine and C-reactive protein levels dropped gradually. The patient was eventually referred to the surgical unit for further management regarding the suspected intra-abdominal abnormality.
Bacteriology. The bacteriological investigations were negative except for the blood cultures. Two sets of aerobic and anaerobic blood culture bottles comprising a total of four bottles were collected during a febrile episode on two separate occasions. Upon arrival at the laboratory, the four bottles were incubated in the BacT/ALERT 3D blood culture system (bioMerieux, Inc., Durham, N.C.). After 48 h of incubation at 35°C, growth in the two anaerobic bottles was detected by the system. Upon Gram staining and direct microscopy from the positive blood culture bottles, straight or slightly curved gram-positive bacilli with a tendency to branching could be observed. Subcultures were made on 5% horse blood agar plates (Svenska LABFAB, Ljusne, Sweden) and incubated aerobically and anaerobically at 37°C. After 2 to 3 days of incubation, visible growth of two morphologically distinct colonies was apparent on the blood agar plates incubated anaerobically (and none on the aerobically incubated plates). The two isolates were similar with regard to their Gram staining features but differed in their colony morphology, colony size, biochemical characteristics, and antibiograms (the zone diameters of clindamycin and imipenem for the two isolates were clearly distinct). A summary of the phenotypic differences between the two isolates is presented in Table 1, and a photograph of the growth of the two isolates on anaerobic blood culture plates is presented in Fig. 1. Further typing of the two isolates with a RapID ANA II identification kit (Remel, Inc., Lenexa, Kans.) was not satisfactory, because the codes suggested for both isolates were not included in the database.
16S rRNA gene sequencing. Species identification of both isolates was carried out by seminested solid-phase sequencing of the bacterial 16S rRNA gene. A first amplification step was performed with primers complementary to the U1 and U8 regions. The amplified products were then used in a second PCR step to amplify the region between U2 and U8 (3). The general PCR primers and the sequencing primers used are presented in Table 2. The PCRs were performed in a GeneAmp 9600 thermal cycler (Applied Biosystems, Perkin-Elmer Europe B.V., Stockholm, Sweden). The amplification products were visualized by agarose gel electrophoresis and ethidium bromide staining. The PCR products were then purified using a Jet Quick Spin Column kit (Genomed GmbH, Germany), and the purified products were processed for DNA sequencing (ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit and ABI Prism 310 Genetic Analyser; Applied Biosystems, Stockholm, Sweden). The sequences, which comprised about 1,100 nucleotides, were then analyzed using Sequencher 3.0 software (Gene Codes Corporation, Ann Arbor, MI). A BLAST search for sequences in GenBank gave 100% agreement with Actinobaculum schaalii (accession number AF487680) for the first isolate and 99.91% agreement (one nucleotide difference) with Actinobaculum urinale (accession number AJ439453) for the second isolate. The phylogenetic relatedness of the isolated Actinobaculum strains to the seven closest matches by BLAST search is presented in Fig. 2.
Discussion. The genus Actinobaculum (gen. nov., 1997) comprises four species at present. Members of the genus, which is phylogenetically related to the genera Actinomyces and Arcanobacterium, are characterized as anaerobic or facultatively anaerobic gram-positive rods (with a tendency to branching), nonmotile, non-spore-forming, and catalase negative. The type species of the genus, Actinobaculum suis (comb. nov., 1997), initially described as Eubacterium suis (9), was classified first as Actinomyces suis in 1992 (5) and then reclassified in 1997 by Lawson et al. (4) in the new genus Actinobaculum. This organism has been isolated from swine and is believed to cause porcine cystitis (8) and abortion (10).
The other three species have rarely been isolated from human sources but are believed to be human pathogens. They include Actinobaculum schaalii (sp. nov., 1997), which was first isolated from the blood of a 64-year-old man with chronic pyelonephritis (4) and in a second case was isolated from the urine of a child with pyeloureteral junction obstruction (6). The species Actinobaculum urinale (sp. nov., 2003) was first isolated from human urine by Hall et al. (2), while the third human pathogen, "Actinobaculum massiliae" (proposed sp. nov., 2002; name is not validly published), was isolated first from the urine of an elderly woman with recurrent catheter-associated cystitis (1) and recently in a case of superficial skin infection (7).
We report on the isolation of two species of Actinobaculum, namely, A. schaalii and A. urinale, from the blood of an elderly patient with a known history of chronic renal insufficiency which was aggravated by a bout of acute diarrhea. This finding undoubtedly raises the suspicion that these organisms may give rise to sepsis, despite the fact that previous similar reports are lacking. Nevertheless, it was difficult in our case to decide whether the deterioration in the patient's renal function could be ascribed to the diarrheal episode per se, to the septicemia caused by the two Actinobaculum species, or to both factors combined. It was not possible either to associate the chronic renal insufficiency observed in this patient with the isolation of these organisms from the patient's blood. Suggestively, a possible urinary tract infection caused by these organisms might have evolved to sepsis. Unfortunately, the urine cultures which were submitted concomitantly with blood cultures were cultured aerobically and thus were negative for Actinobaculum as well as for other urinary pathogens. It should be noted in this context, however, that due to the anaerobic growth characteristics of these organisms and their slow growth, they are often missed in routine urine cultures, and infections by these organisms may thus be underdiagnosed. It is suggested, therefore, that special culture procedures be applied whenever infection by such organisms is suspected. No further blood cultures were submitted to the laboratory after the antibiotic therapy, because the patient made an uneventful recovery from the acute febrile episode.
It has been stated by some authors that some strains belonging to Actinobaculum may exhibit phenotypic heterogeneity of the colonies after 5 days of incubation. This may lead to the erroneous interpretation of sample contamination with two different species of gram-positive bacilli or, alternatively, a false impression of double infection by two species of Actinobaculum when in fact only one species is present (1). In this case it was apparent from the beginning that the two isolates obtained in pure culture were distinct species based on the fact that they were different in their colonial morphological appearances and growth characteristics, prompting the laboratory to proceed with the identification of both variants. The urease test performed on both isolates was also discriminatory, because A. urinale is urease positive, while A. schaalii is urease negative. Furthermore, the discrepancy in the zone diameters of clindamycin and imipenem was obvious. These findings were subsequently confirmed by the 16S rRNA sequencing of the two isolates.
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