Isolation of Robinsoniella peoriensis from Four Human Specimens

Abstract

Robinsoniella peoriensis is a recently described anaerobic, spore-forming, Gram-positive bacillus originally recovered from swine manure. We report four human cases in which R. peoriensis was isolated from clinical samples.

CASE REPORTS

Between February 2009 and January 2010, four clinical isolates of Gram-positive, anaerobic, spore-forming bacilli from different hospitals in the United States received by the Clinical Microbiology Laboratory at the Mayo Clinic, Rochester, MN, were identified as Robinsoniella peoriensis by 16S rRNA gene sequencing. Clinical information was provided by the patients' health care providers.

Case 1.

A 61-year-old woman underwent laparoscopic sigmoid colectomy due to diverticulitis. The procedure was complicated by a left-sided fluid collection, which was aspirated by interventional radiology. Bacterial, mycobacterial, and fungal cultures of the aspirated fluid were negative. Treatment with moxifloxacin and clindamycin was empirically started. However, she developed fever and abdominal pain and was admitted to the hospital. Piperacillin-tazobactam and vancomycin were empirically started, and the patient underwent percutaneous drainage of a purulent left-sided abdominal fluid collection by interventional radiology. A Gram-stained smear of the fluid revealed numerous polymorphonuclear leukocytes and a few small Gram-positive bacilli. Bacterial cultures yielded Gram-positive coccobacillary organisms from thioglycolate broth on day 4 and from a CDC anaerobic plate on day 7 of incubation. The organism was sent to the Mayo Clinic for identification. The patient's clinical condition improved with drainage of the fluid collection and vancomycin and piperacillin-tazobactam administration.

Case 2.

A 68-year-old woman with no significant past medical history was admitted to the hospital after being struck by an automobile. She sustained open pelvic and femur fractures with gross soil contamination and underwent surgical wound debridement. A direct Gram-stained smear of the intraoperative samples showed many polymorphonuclear leukocytes and Gram-negative rods. Cultures of surgical tissue revealed Acinetobacter sp., Enterococcus sp., and a Gram-negative anaerobic bacillus. The anaerobic bacillus was isolated on Brucella agar after 48 h of incubation and was referred to the Mayo Clinic for identification. After multiple debridements and broad-spectrum antibiotic treatment, the patient recovered without complications.

Case 3.

A 45-year-old man with no significant past medical history was admitted to the hospital after a motor vehicle accident with right tibial and fibular fractures, which were treated by open reduction and internal fixation. One month after the surgical procedure, foul-smelling drainage from the surgical wound was noted and debridement of the wound was performed. Intraoperative cultures grew vancomycin-resistant Enterococcus faecium, and linezolid was administrated for 6 weeks. Soon after the end of the antibiotic treatment, recurrent wound drainage was noted and surgical debridement and removal of the fixation hardware were performed. Gram-stained smears of the intraoperative specimens revealed polymorphonuclear white blood cells but no organisms, and anaerobic cultures grew rare smooth nonhemolytic colonies on CDC anaerobic agar after 48 h of incubation. A Gram stain showed a Gram-positive bacillus with subterminal spores resembling Clostridium spp. The RapID ANA II system (Remel, Lenexa, KS) identified the organism as Clostridium tertium (99.9%) but with a questionable level of confidence due to rare biofrequency. The isolate was referred to the Mayo Clinic for identification. The patient was treated with clindamycin but 4 weeks later had a recurrence of the wound drainage, and another surgical debridement was performed. Intraoperative cultures grew a Gram-positive, spore-forming, anaerobic bacillus that could not be identified by the RapID ANA II system. It was assumed to be the same organism which had been referred to the Mayo Clinic. Clindamycin was continued and linezolid was added for 6 weeks of treatment; the leg wound healed without any further sign of infection.

Case 4.

A 79-year-old woman with a history of diabetes, hypertension, and hypercholesterolemia underwent coronary artery bypass surgery for myocardial infarction. While in the operating room, the patient had a cerebrovascular accident with residual left hemiparesis. She was transferred to a rehabilitation facility and subsequently developed diarrhea and altered mental status. She was referred to an emergency department, where she was found hypotensive and tachycardic. Her white blood cell count was 25,000/μl with 86% neutrophils. A Clostridium difficile toxin assay was negative, and a urine analysis and a urine culture showed pyuria and Proteus mirabilis, respectively. Two sets of BacT/Alert system (bioMérieux, Durham, NC) blood cultures were obtained. One out of four blood culture bottles signaled positive on day 3 of incubation. This isolated anaerobic Gram-positive bacillus was referred to the Mayo Clinic for identification. Piperacillin-tazobactam, levofloxacin, and metronidazole were empirically started, but the patient suffered a cardiac arrest and died 96 h after admission.

Microbiology methods.

All isolates received at the Clinical Microbiology Laboratory of the Mayo Clinic were subcultured to CDC sheep blood agar and incubated in an anaerobic chamber (Anaerobic Glove Box; Coy Laboratory Products Inc., Ann Arbor, MI) at 35°C. A chocolate blood agar plate was streaked and incubated in 5% CO₂ at 35°C to assess aerotolerance. Small, smooth, nonhemolytic colonies were seen after 48 h of incubation on CDC sheep blood (Fig. 1). Gram-stained smears showed Gram-positive bacilli with subterminal spores that did not cause swelling of the cells (Fig. 2). The isolates were identified using 16S rRNA gene sequencing. DNA was prepared for amplification using PrepMan Ultra (Applied Biosystems, Foster City, CA), and subsequently the DNA was amplified and sequenced with 16S rRNA gene universal primers (5) using the BigDye terminator method by the Veriti 96-Well Fast thermal cycler (Applied Biosystems, Foster City, CA) and detected by the 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequences generated (477 bp) were compared against the Mayo Anaerobe database and the MicroSeq 500 database library. However, no satisfactory match was found in these databases. The sequences were then compared to those in the GenBank database, showing 99.7, 99.7, 99.4, and 99.4% identity with R. peoriensis (GenBank accession no. AF445288 and DQ681227.1) for cases 1 to 4, respectively. Blautia coccoides (cases 1 to 3) and Ruminococcus productus (case 4) were the next closest matches, showing 95.8, 94.9, 95.3, and 95.3% identity with the patients' sequences. However, as of 27 June 2010, the sequences of our isolates also showed between 99.5 and 99.7% identity with other sequences in the GenBank database (accession no. AF445256.1, AF445288.1, AF445197.1, AF445258.2, AF4452871, AF445257.1, AF445286.1, and AF445255.1). These sequences correspond to other bacteria isolated from swine manure which are not yet fully characterized and could represent related species of Robinsoniella.

FIG. 1.

CDC blood agar after 48 h of incubation showing nonhemolytic smooth colonies of R. peoriensis.

FIG. 2.

Gram-stained smear showing R. peoriensis. Gram-positive bacilli with subterminal spores are visible.

For further characterization, three of the isolates (from cases 1, 2, and 4) were subjected to biochemical testing and analysis of volatile and nonvolatile fatty acid end products by gas-liquid chromatography using an Agilent 6850 gas-liquid chromatograph (Agilent, Wilmington, DE) (Table 1). These three isolates were also tested using the RapID ANA II system (Remel, Lenexa, KS) for identification. Two isolates were identified as C. tertium with a questionable level of confidence due to rare biofrequency. One isolate was identified as Actinomyces israelii or Clostridium perfringens with a questionable level of confidence due to rare biofrequency.

TABLE 1.

Biochemical characteristics of R. peoriensis clinical isolates in this study

Biochemical reaction	This study (%)a	R. peoriensis (PPC31)a,b	C. tertium (6)a
Colistin disc (10 μg)	Resistant (100)	NAc	NA
Vancomycin disc (5 μg)	Susceptible (100)	NA	Susceptible
Indole	− (100)	−	−
Catalase (15%)	− (100)	NA	−
Motility	− (100)	−	+
Egg yolk agar	Growth (100)	NA	−
Lecithinase	− (100)	NA	−
Lipase	− (100)	NA	−
Fluorescence	− (100)	NA	NA
Esculin hydrolysis	+ (100)	NA	+
Gelatin	− (100)	NA	−
Starch hydrolysis	− (100)	+	+
Urea	− (100)	−	−
Acid from:
Glucose	+ (100)	+	+
Fructose	+ (100)	+	+
Glycogen	+ (66)	NA	NA
Galactose	+ (100)	NA	NA
Lactose	+ (100)	+	+
Maltose	+ (100)	+	+
Mannitol	− (100)	NA	+
Mannose	+ (100)	−	+
Melibiose	+ (100)	NA	+
Ribose	+ (100)	NA	+
Salicin	+ (100)	NA	+
Sorbitol	+ (66)	NA	NA
Starch	+ (66)	NA	NA
Sucrose	+ (100)	NA	+
Xylose	+ (100)	+	Variable
Metabolic end productsd	A, p, l, s	A, s, f, l	A, B, L

^a−, negative; +, positive.

^bType strain.

^cNA, not available.

^dA, acetic acid; B, butyric acid; p, propionic acid; l, lactic acid; s, succinic acid; f, formic acid. Capital letters indicate major production.

Antimicrobial susceptibility testing was performed on three isolates (from cases 1, 2, and 4) using the Etest (AB bioMérieux, Marcy l'Etoile, France) according to the manufacturer's instructions. The isolates were resistant to penicillin (MICs, 6 to 8 μg/ml) and clindamycin (MICs, 8 to 12 μg/ml) but susceptible to piperacillin-tazobactam (MICs, 8/4 to 16/4 μg/ml), ertapenem (MICs, 0.75 to 1.0 μg/ml), and metronidazole (MICs, 0.094 to 0.5 μg/ml). All isolates were negative for beta-lactamase production by the nitrocefin disc assay.

Discussion.

As part of a microbiological survey of swine manure, five unknown isolates of anaerobic, spore-forming, Gram-positive bacilli were recovered (3). Six years after the isolation of these unknown Gram-positive anaerobes, a representative strain was phenotypically and phylogenetically characterized (2). It was determined that this isolate belonged to the phylum Firmicutes within the family Lachnospiraceae. Phylogenetic analysis showed that this organism belonged to the clostridial rRNA cluster XIVa subgroup, with the closest related genus being Ruminococcus. The organism was named R. peoriensis. Cotta et al. described R. peoriensis as an oval-to-rod-shaped bacterium with subterminal spores that grows in 5% CO₂ or under anaerobic conditions at 37°C, although with our isolates the aerotolerance test was negative. It produces small, nonhemolytic colonies (0.5 to 1.5 mm) and ferments glucose, lactose, and maltose. It does not reduce nitrate or produce indole.

Concurrently with the initial description of R. peoriensis, a Gram-positive bacillus with a similar biochemical profile was isolated from the deep heel wound of a 79-year-old woman in Sweden (2). More recently, R. peoriensis was isolated from blood cultures of a patient with pancreatic cancer (7). The most common source of anaerobic infections is endogenous normal flora or environmental contamination of wound injuries (1, 4). Two of our patients developed soft-tissue infections with R. peoriensis after traumatic injuries. As R. peoriensis was isolated from swine manure, it is possible that it could also colonize the gastrointestinal tracts of other animals, resulting in soil contamination. Although this anaerobic bacillus has not yet been isolated as normal flora from humans, the presentation of case 1 suggests that the source of infection was the disruption of the mucosal barrier. In case 4, R. peoriensis was isolated from one of four blood culture bottles; the pathogenic role of this organism in this patient is unclear. Whether or not her diarrhea was associated with transient R. peoriensis bacteremia is unknown.

Susceptibility testing showed penicillin and clindamycin resistance but susceptibility to piperacillin-tazobactam and metronidazole. It should be noted that although the isolate from patient 3 was never tested for antimicrobial susceptibility, based on the other isolates, use of clindamycin in this case could have contributed to treatment failure.

In summary, we report four cases in which R. peoriensis was isolated from human specimens. Identification of R. peoriensis might be challenging in clinical laboratories without access to molecular methods, as commercial identification systems may not identify or may misidentify this organism.