INTRODUCTION

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Anaerobiospirillum spp. consist of a group of spiral, motile, gram-negative, anaerobic rods that have been isolated from the feces of mammals including cats, dogs, and humans (2, 10). Anaerobiospirillum succiniciproducens has been implicated as a rare cause of diarrheal illness and bacteremia in humans (7, 10, 11, 14, 19-22, 25). Previous reports have shown that the infection is usually associated with or originates from the gastrointestinal tract (10, 15).

In this report, we describe three cases of A. succiniciproducens bacteremia, including the first report of A. succiniciproducens infection in a human immunodeficiency virus (HIV)-infected individual. In addition, a search of the sequence databases did not reveal the 16S rRNA gene sequence of A. succiniciproducens. We sequenced the 16S rRNA genes of the type strain, strain ATCC 29305, and the three Australian clinical isolates and present the phenotypic and genotypic characteristics of these three isolates.

	CASE REPORTS

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Patient 1. A 53-year-old man with known alcoholic liver disease presented with epigastric pain, hematemesis, and melena. Examination revealed hypotension, hepatomegaly, and peripheral signs of chronic liver disease. On admission, full blood examination, serum urea levels, and electrolyte levels were normal. He was transfused with 3 units of packed erythrocytes. Esophagogastroscopy revealed bleeding esophageal varices which were treated by injection sclerotherapy. On the following day he became febrile (temperature, 38.2°C). There were no associated symptoms; in particular, there was no diarrhea. Blood was taken for culture. He received no antibiotics, subsequently became afebrile, and was discharged 2 days later.

Patient 2. A 48-year-old homosexual man with advanced AIDS (20 CD4 cells/mm³) was known to have been HIV antibody positive since 1989. Previous AIDS-defining illnesses included a low-grade B-cell gastric non-Hodgkin's lymphoma successfully treated with combination cytotoxic chemotherapy in 1992 and, in 1994, cytomegalovirus retinitis treated with induction and ongoing maintenance ganciclovir. In February 1996, a biopsy of an axillary lymph node revealed a T-cell non-Hodgkin's lymphoma. Bone marrow involvement was demonstrated, and he received a course of oral etoposide and prednisolone. In August 1996, endoscopy revealed multiple gastric ulcers that on biopsy demonstrated lymphomatous infiltration, and a further course of oral chemotherapy was commenced. Two weeks after chemotherapy he was admitted with lethargy, weight loss, leg weakness, and urinary frequency related to HIV myelopathy. After 4 days in hospital he developed high swinging fevers (up to 38.7°C), a productive cough, and diarrhea. Full blood examination revealed a hemoglobin concentration of 89 g/liter, a leukocyte count of 2.99 × 10⁹/liter, and a platelet count of 160 × 10⁹/liter. On the blood film, the neutrophils showed a left shift, toxic granulations, vacuolation, and Dohle bodies. Serum electrolyte levels and liver function test results were normal. Chest X rays revealed bilateral lower-zone interstitial changes. Blood was collected for culture. Cultures of cerebrospinal fluid, sputum, and urine were negative. Cultures of blood for mycobacteria were negative. Fecal specimens were not collected. Intravenous treatment with ticarcillin-clavulanate at a dosage of 3.1 g every 6 h was commenced empirically; however, the patient continued to deteriorate and died 7 days later. A request for a postmortem examination was declined.

Patient 3. A 57-year-old man with a 3-year history of non-Hodgkin's lymphoma presented 12 days postchemotherapy complaining of a 3-day history of fever and night sweats, a 2-week history of malaise, and a long-standing history of a nonproductive cough. One year prior to admission he had had a splenectomy and had been receiving penicillin prophylaxis. On examination he was found to be febrile (temperature, 38°C), but he had no localizing signs. An echocardiogram was performed because of aortic stenosis, but no abnormalities were detected. Three sets of blood cultures were prepared. Full blood examination revealed a raised leukocyte count with a predominant lymphocytosis with circulating lymphoma cells and a marked neutropenia attributed to recent chemotherapy. The patient was empirically treated intravenously with ticarcillin-clavulanate and gentamicin for 4 days and then orally with ciprofloxacin for 3 days, with rapid resolution of his symptoms.

	MATERIALS AND METHODS

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Organism isolation and phenotypic characterization. The BACTEC Blood Culture System was used for patients 1 and 3. The blood of patient 1 was inoculated into a set of BACTEC bottles (6D and 7D bottles; Johnston Laboratories Inc., Cockeysville, Md.), and BACTEC Fluorescent Aerobic and Anaerobic Resin bottles were used to culture the blood of patient 3. The blood of patient 2 was inoculated into one BacT/Alert aerobic FAN bottle and one BacT/Alert anaerobic bottle (Organon Teknika Corporation, Durham, N.C.). The contents of the bottles were subcultured onto 6% horse blood agar, and the plates were incubated at 37°C for 2 days under anaerobic conditions.

Antibiotic susceptibility testing. The isolates were tested for their susceptibilities to ampicillin-clavulanate, chloramphenicol, ciprofloxacin, clindamycin, imipenem, metronidazole, penicillin, and ticarcillin-clavulanate by the Epsilometer test (Etest; AB Biodisc, Solna, Sweden) according to the manufacturer's recommended guidelines (1). The medium used was brucella agar plus 5% defibrinated sheep blood and 1 mg of vitamin K1 per liter (final concentration). A suspension of organism with a turbidity equivalent to that of a no. 1 McFarland standard was used, and the MICs were read after 48 h of incubation under anaerobic conditions. One of the recommended quality control organisms, Bacteroides fragilis ATCC 25285, was also tested (16).

DNA extraction and purification, PCR, and 16S rRNA gene sequencing. Whole-cell chromosomal DNA was extracted and purified from plate-grown bacteria by the cetyltrimethylammonium bromide method as described previously (24).

rRNA sequence alignment and phylogenic tree construction. The complete 16S rRNA gene sequence was compared with other known rRNA gene sequences in the GenBank database and the Ribosome Database Project (RDP) database (University of Illinois, Urbana). The complete 16S rRNA gene sequence was aligned by using the aligned database of the small-subunit rRNA sequences in the RDP database (9). Positions with gaps or uncertain bases were removed, and phylogenetic trees were constructed with the PHYLIP package (3) and the maximum-likelihood program fDNAml (18) implemented by the Australian National Genomic Information Service.

Nucleotide sequence accession number. The sequence of the type strain of A. succiniciproducens, strain ATCC 29305, has been lodged with the GenBank database, and the accession no. is U96412.

	RESULTS

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The isolate from patient 1 was recovered from the BACTEC 7D anaerobic bottle after 5 days of incubation. The strain from patient 2 was isolated from the BacT/Alert anaerobic bottle (Organon Teknika) after 4 days of incubation. The strain from patient 3 was recovered from a BACTEC Fluorescent Anaerobic Resin bottle after 2 days of incubation. Positive signals were noted for all three blood culture systems. Gram-negative spiral organisms were seen on Gram smears prepared with samples from the anaerobic blood culture bottles for all patients. Under anaerobic conditions, growth was detected after 48 h of incubation at 37°C but not at 25 or 42°C. No growth occurred under aerobic or microaerobic conditions.

After 48 h the colonies on blood agar were 0.5 to 1 mm in diameter, translucent, and nonhemolytic, with some colonies showing feathery swarming growth. Phase-contrast microscopy revealed spiral-shaped organisms exhibiting a corkscrew-like motility but without the typical rapid darting motility of Campylobacter spp. Gram-stained smears revealed gram-negative spiral cells 0.6 to 0.8 m wide and 4.0 to 8.0 m long (Fig. 1). Upon repeated subculture, there was an increased amount of straight rod-shaped organisms. An electron micrograph revealed a curved bacterium with bipolar tufts of flagella. Staining of the flagella performed with a colony from a plate after 2 days of growth also showed spiral bacteria with polar multitrichous flagella, some with distinctive bipolar tufts (Fig. 2).

View larger version (189K): [in this window] [in a new window]   FIG. 1.   Light microscopy photomicrograph of a Gram-stained smear of a culture of A. succiniciproducens showing characteristic spiral forms. Magnification, ×1,000.

View larger version (178K): [in this window] [in a new window]   FIG. 2.   Light microscopy photomicrograph of a 48-h culture of A. succiniciproducens showing bacteria with polar multitrichous or tufted flagella. Magnification, ×1,000.

Biochemical tests performed with these isolates showed that reactions for catalase, oxidase, indole, and nitrate reduction were all negative. The API-ZYM strips detected the presence of activity for the enzymes leucine arylamidase, -galactosidase, -glucosidase, and N-acetyl--glucosaminidase for ATCC 29305 and the three clinical isolates. The positive tests with the Rapid ANA II system were for o-nitrophenyl--D-galactopyranoside (-galactosidase), -glucosidase, glycine aminopeptidase, arginine aminopeptidase, and serine aminopeptidase for all four isolates. A profile or microcode of 030441 was obtained.

Gas-liquid chromatography revealed that acetic acid is the major volatile fatty acid that was produced and that succinic acid is the major nonvolatile acid produced from glucose cooked-meat medium.

Table 1 presents the MICs for the clinical isolates and the type strain of A. succiniciproducens, strain ATCC 29305, obtained by the Etest. All isolates were resistant to clindamycin but sensitive to chloramphenicol, imipenem, amoxicillin-clavulanate, and ticarcillin-clavulanate. The isolates from patients 1 and 3 were resistant to metronidazole. The isolate from patient 1 was resistant to penicillin. Although there are no documented breakpoints for ciprofloxacin for anaerobic organisms, for all three isolates MICs were in the susceptible range for aerobic bacteria (17).

The complete 16S rRNA gene sequences of the three clinical isolates and type strain ATCC 29305 were determined and compared with all known 16S rRNA sequences found in the sequence databases (RPD and GenBank). The sequences of the isolates from patients 1 and 3 were identical to that of type strain ATCC 29305 across the entire lengths of the 16S rRNA genes, but the sequence of the isolate from patient 2 differed at 4 bases (T instead of C at position 188, A replacing C at position 190, T replacing C at position 597, and A replacing T at position 792). The sequence of type strain ATCC 29035 was found to be most similar to that of the species Ruminobacter amylophilus, with 88.3% sequence similarity. Phylogenetic trees were inferred by a previously described method (23), and a representative example is presented in Fig. 3. Maximum-likelihood (fDNAml), distance matrix, and parsimony (PAUP) methods all gave very similar branching orders. Bootstrap analyses strongly supported the branching of A. succiniciproducens within the gamma subdivision of the proteobacteria, with R. amylophilus being the closest relative.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Inferred phylogeny of the proteobacteria, obtained by using complete 16S rRNA gene sequences, showing the relationship of A. succiniciproducens to its closest relatives. Note that Haemophilus actinomycetemcomitans is validly named as Actinobacillus actinomycetemcomitans, but it retains the genus name Haemophilus at the American Type Culture Collection web site and this usage is consistent with the GenBank entry for this sequence. The tree shown was produced by the maximum-likelihood (fDNAml) method, but distance matrix (PHYLIP) and parsimony (PAUP) methods gave very similar branching orders. Bootstrap values were derived by distance matrix and parsimony methods (values above and below the nodes, respectively), and only the significant bootstrap values are shown. B. subtilis was used as the outgroup. The scale bar represents 0.1 expected nucleotide substitution per site.

	DISCUSSION

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A. succiniciproducens was first isolated in 1976 by Davis et al. (2) from the throats and cecal contents of beagle dogs. Recently, another member, Anaerobiospirillum thomasii (12), was proposed to include a previously described group of Anaerobiospirillum-like organisms isolated from feces of mammals. To our knowledge, Anaerobiospirillum-like organisms or A. thomasii has not been reported to be associated with bacteremia in humans. A. succiniciproducens and A. thomasii can be differentiated by biochemical tests with carbohydrate fermentations and by the enzyme profiles obtained with API-ZYM strips, notably, the absence of -galactosidase and -glucosidase activities (12).

A. succiniciproducens is a rare cause of human infection. In a review of infections involving uncommonly encountered motile anaerobic gram-negative bacilli at the Veterans Administration (now the U.S. Department of Veterans Affairs) Wadsworth Medical Center from 1973 to 1985, an Anaerobiospirillum sp. was not isolated (5). A study conducted by the Centers for Disease Control and Prevention (CDC), Atlanta, Ga., reviewed 22 patients (including 4 patients who had been described previously) with A. succiniciproducens bacteremia whose isolates were submitted from 1975 to 1986. While most cases of A. succiniciproducens bacteremia described in the literature occurred in the United States (15, 20, 21), more recent reports from Hong Kong (19), South Africa (14), New Zealand (4), and now Australia suggest that this organism is more prevalent than was previously thought and may occur across wider geographical regions. Better methods of detection by blood culture with automated systems such as the BACTEC Blood Culture System and the BacT/Alert Blood Culture System in laboratories and the increased awareness of the importance of this organism as a cause of human disease may have been factors contributing to the increase in the rate of isolation and detection of this unusual organism.

Previous reports (7, 10, 11, 14, 19-22, 25) have indicated that Anaerobiospirillum spp. are an uncommon cause of gastroenteritis and bacteremia. Most patients with diarrheal disease did not have an underlying disorder, and most patients recovered from the infection (10, 13). In contrast, almost all reported cases of bacteremia (15, 20-22) have been in patients with an underlying illness such as alcoholism, malignancy, diabetes mellitus, and other gastrointestinal disorders. The three patients reported in our study all had underlying disease: one had alcohol-related liver disease complicated by gastrointestinal hemorrhage, one had non-Hodgkin's lymphoma, and the other had advanced AIDS. This suggests that immunosuppressed or immunocompromised hosts may be more susceptible to infection with this organism.

The gastrointestinal tract has been postulated as a likely portal of entry in patients with A. succiniciproducens bacteremia. McNeil et al. (15) reported that 77% of 22 patients with bacteremia had gastrointestinal signs or symptoms, with gastrointestinal bleeding being a feature in 36% of patients, and that on microscopic examination spiral organisms were seen in the feces of some patients. Malnick et al. (10) screened feces for Anaerobiospirillum spp. but failed to detect the organisms in 527 healthy, asymptomatic human subjects but did detect them in 7 of 10 cats and 3 of 10 dogs. This suggests that Anaerobiospirillum spp. are unlikely to be part of the normal gastrointestinal flora of humans. They reported the isolation of A. succiniciproducens or Anaerobiospirillum-like organisms from the feces of 17 humans with diarrhea. Of these 17 patients, detailed information was obtained for only 5 patients, all of whom had pet dogs, which raises the possibility of zoonotic transmission. The source of infection in our three patients was unclear. Gastrointestinal symptoms were absent in our non-HIV-infected individuals, and fecal specimens were not collected from our HIV-infected patient, despite the presence of diarrhea. For our three patients, information was unavailable about contact with domestic animals such as dogs or cats.

Antimicrobial susceptibility testing by various investigators (5, 15) has shown that clinical isolates of A. succiniciproducens are susceptible to carbenicillin, chloramphenicol, and cephalothin and are resistant to vancomycin and nalidixic acid. Susceptibilities to penicillin G, ampicillin, erythromycin, clindamycin, and metronidazole were variable. Although 16 patients in a study by McNeil et al. (15) received antimicrobial treatment, the effect on the outcome of the infection was unclear. For seven of those patients bacteremia was reported to have contributed to their deaths. In the current study, the three isolates were found to be sensitive to chloramphenicol, ciprofloxacin, and imipenem but resistant to clindamycin. Two of the three isolates was also resistant to metronidazole. Because clindamycin and metronidazole are first-line antibiotics for the treatment of infections caused by anaerobic bacteria, susceptibility testing of all isolates is advisable. For all three isolates the MICs to penicillin were increased, and the isolate from patient 1 was found to be resistant to penicillin. Because high levels of penicillin are achievable in blood, infection with isolates for which MICs are of this order may be treatable. Our first patient recovered spontaneously, even though he received no antibiotics. In contrast, the HIV-infected patient died at 7 days, despite intravenous treatment with ticarcillin-clavulanate. The third patient received ticarcillin-clavulanate, gentamicin, and ciprofloxacin, with rapid resolution of symptoms.

Anaerobiospirillum spp. were often erroneously identified as Campylobacter (11, 14, 20, 25) because of similar Gram staining morphologies and corkscrew-like motilities. Demonstration of polar tufts of flagella on a stained smear preparation is crucial in differentiating Anaerobiospirillum from closely related genera. However, not all laboratories have electron microscopy facilities readily available for use for the identification of microbes. In contrast, staining of the flagella by the method described by Kodaka et al. (6) is simple and does not require special equipment. Biochemical tests with commercial kits such as the Rapid ANA II system and Oxoid ANident discs were unhelpful in the identification of this organism because Anaerobiospirillum spp. were not included in the databases provided with those kits. The availability of the 16S rRNA gene sequence will allow more definitive and accurate identification and classification of Anaerobiospirillum isolates.