Identification by 16S rRNA Gene Sequencing of Negativicoccus ...

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 2011, p. 3082–3084 0095-1137/11/$12.00 doi:10.1128/JCM.01913-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 49, No. 8

Identification by 16S rRNA Gene Sequencing of Negativicoccus succinicivorans Recovered from the Blood of a Patient with Hemochromatosis and Pancreatitis䌤 D. L. Church,1,2,3* K. E. Simmon,4 Jan Sporina,3 T. Lloyd,1 and D. B. Gregson1,2,3 Calgary Laboratory Services (CLS)1 and Departments of Pathology & Laboratory Medicine2 and Medicine,3 University of Calgary, Calgary, Alberta, Canada, and Associated Regional and University Pathologists Laboratories (ARUP), Salt Lake City, Utah4 Received 20 September 2010/Returned for modification 10 November 2010/Accepted 26 May 2011

We describe a case of Negativicoccus succinicivorans bacteremia in an adult man with hemochromatosis and acute pancreatitis. Conventional phenotypic tests and commercial identification systems failed to definitively identify the tiny anaerobic Gram-negative coccus isolated from two sets of blood cultures. The bacterium was identified by 16S rRNA gene sequencing and analysis using the SmartGene Integrated Database Network System software. This is the first published report of the recovery of this organism from a patient with invasive infection.

Microbiological data. The anaerobic (FN) BacT/Alert blood culture bottles were positive for Gram-negative coccoid bacteria at 17.9 h in one set and at 28 h in the second set. A Gram stain of the blood pellet showed Gram-negative cocci. An acridine orange stain also confirmed the presence of cocci. Blood was subcultured to Columbia blood agar (BA), chocolate agar (CHOC), MacConkey agar (MAC), and Brucella blood agar (BBA) (PML Microbiologicals, Wilsonville, OR) plates and incubated anaerobically at 35°C for 48 h before examination. No growth occurred on the BA, CHOC, or MAC plates, even with extended incubation under the same conditions for another 24 h (i.e., a total of 72 h). Dusty growth of tiny translucent colonies (colonies of ⬍0.5 mm in diameter) occurred on the BBA plate after 48 h of anaerobic incubation at 35°C using an Anoxomat Mark II system (Mark Microbiology, Drachten, Netherlands). The isolate did not grow under microaerophilic conditions. An acridine orange stain of the colony showed cocci, while a Gram stain showed the isolate to be a Gramnegative coccus. Disk testing showed the isolate to be resistant to vancomycin (5 ␮g), colistin (10 ␮g), and metronidazole (5 ␮g) but sensitive to kanamycin (500 ␮g). The isolate was also found to be bile susceptible by a 1-␮g disk test. Biochemical analysis using the Vitek 2 ANC card (bioMe´rieux, Laval, Quebec, Canada) gave a phenotypic identification as a Veillonella species (97% probability). The isolate was inert, except for a positive L-pyrrolidonyl-arylamidase (PyrA) reaction. Metabolic end products were not tested. Antibiotic susceptibility testing using Etest strips (AB BioMe´rieux, Laval, Quebec, Canada) showed the isolate to be sensitive to penicillin (MIC ⫽ 0.2 ␮g/ml) but resistant to clindamycin (MIC ⬎ 256 ␮g/ml) and metronidazole (MIC ⬎ 256 ␮g/ml). Molecular identification was done by partial sequencing of the 16S rRNA gene with MicroSeq 500 kits and an ABI Prism 3100 sequencer (Applied Biosystems, Foster City, CA) according to standard methods (1). A BLAST search of the GenBank database gave no definitive identification other than “uncultured bacterium.” A BLAST search against the SmartGene Integrated Database Network System (IDNS) bacterial

CASE REPORT A 57-year-old man with hemochromatosis was admitted to the hospital in June 2008 with acute pancreatitis. He had a history of chronic alcohol abuse. He complained of abdominal pain and diarrhea. The physical examination revealed a temperature of 38.4°C and lower abdominal tenderness without rebound tenderness. Computerized tomography scanning revealed peripancreatic inflammation consistent with pancreatitis. A complete blood count and differential showed anemia (hemoglobulin ⫽ 118 g/liter), thrombocytopenia (68 ⫻ 109 cells/ml), and a normal white blood cell count (6.9 ⫻ 109 cells/ml), with a normal level of polymorphonuclear cells but a low level of lymphocytes. He had mild renal dysfunction, with a serum creatinine level of 111 g/liter. Pancreatitis was confirmed, and he had an increasingly elevated lipase level (from 225 to 978 IU), documented within the initial 48 h in the hospital, and associated mild hepatocellular dysfunction with an elevated gamma-glutamyl transferase level (587 g/liter). The total bilirubin was also mildly increased at 16 g/liter. Stool tests were negative for Clostridium difficile toxins (A and B), Salmonella, Shigella, Campylobacter, Escherichia coli O157, Aeromonas, and Plesiomonas, and a Giardia/Cryptosporidium enzyme immunoassay (EIA) was negative. Two sets of blood samples were collected and cultured with BacT/Alert blood culture system (bioMe´rieux Canada, Laval, Quebec, Canada) FA (aerobic) and FN (anaerobic) bottles, and the FN bottles in both sets were positive, as described below. Supportive therapy included administration of antibiotics (one dose of cefuroxime and then piperacillin-tazobactam), and the patient recovered uneventfully after 4 days of hospitalization and was discharged.

* Corresponding author. Mailing address: Calgary Laboratory Services, 9-3535 Research Rd. N.W., Calgary, Alberta, Canada T2L 2K8. Phone: (403) 770-3281. Fax: (403) 770-3347. E-mail: Deirdre.church @cls.ab.ca. 䌤 Published ahead of print on 8 June 2011. 3082

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FIG. 1. Neighbor-joining tree of 1,314 bp of the 16S rRNA gene sequences of the blood isolates and reference strains. Branch support is recorded at the nodes as a percentage of 1,000 bootstrap iterations.

database indicated that the species most closely related to the blood isolate was Dialister micraerophilus, but the overall identity score was 91.1% and there were more than 30 mismatches (7, 11). The isolate was referred in 2008 to the molecular microbiology laboratory of the Associated Regional University Pathologists (ARUP) at the University of Utah for full nucleic acid sequencing of the 16S rRNA gene (⬃1,500 bp). No definitive identification was obtained from the nearly full-length 16S rRNA gene sequence with either the GenBank or the IDNS database at that time. However, a recent BLAST search (July 2010) of the clinical sequence using both the GenBank and IDNS databases demonstrated references with 100% identity. The references were to a recently described species, Negativicoccus succinicivorans (type strain ADV 07/08/06-B-1388T), within the Veillonellaceae family in the phylum Firmicutes that was recently isolated from human skin and soft tissue samples and described using phenotypic and genetic methods (8). Matching of the complete 16S rRNA gene sequence (⬃1,527 bp) from our clinical bacteremia isolate to the nearly complete reference sequences (⬃1,401 bp) from the skin and soft tissue Negativicoccus type strain from GenBank (accession numbers FJ715928, FJ715929, and FJ715930) using both the GenBank and the IDNS databases showed them to be highly (⬎99%) similar. Similar 16S rRNA gene sequence identities (⬎99%) were also observed with sequences of several uncultured clones from the human skin microbiome (GenBank accession numbers GQ01459 and GQ14745) and with several uncultured clones from human urogenital (vaginal) epithelium (GenBank accession numbers AY958803 and HM330774) (3, 4). Our isolate also showed good identity (⬎90%) with members of the genus Dialister within the family Veillonellaceae (8). The most closely related known species within GenBank was Dialister micraerophilus, with a sequence identity of 91.1%, while other Dialister species sequences within the IDNS database gave a

slightly higher identity (93%) (7). Alignment of the complete 16S rRNA sequence from our clinical bacteremia isolate and three nearly complete 16S rRNA sequences from the Negativicoccus type strain using ClustalW2 (http://www.ebi.ac.uk/Tools /clustalw2/index.html) also showed almost complete identity (99.9%) of these sequences. Phylogenetic analysis using MEGA 4.0 software (12) and the neighbor-joining method also showed high bootstrap scores between our blood isolate and the sequences from the Negativicoccus type strain (8), while other genera in the family Veillonellaceae, Veillonella and Dialister in particular, were closely related but had lower bootstrap scores (Fig. 1).

Discussion. This report demonstrates that partial and complete sequencing of the 16S rRNA gene is a valuable tool for definitive molecular identification of important clinical isolates that cannot be readily identified by phenotypic methods (1, 8, 11). This is the first report of Negativicoccus succinicivorans causing bacteremia from an abdominal source in humans. Because our patient did not have obvious skin compromise, his bacteremia most likely originated from an abdominal source since he had diarrhea, abdominal pain, pancreatitis, and hemochromatosis, which disrupts the integrity of intestinal mucosa and portal circulation. Several genera within the family Veillonellaceae have been isolated from feces, including Veillonella spp. and Megasphaera spp. (2, 6). Veillonella parvula group isolates and Dialister succinatiphilus sp. nov. have also recently been shown to be part of the intestinal flora using molecular analyses (5, 10). Although bacteremia has rarely been described for this group of organisms, Veillonella parvula, in particular, has recently been implicated as a cause of secondary discitis due to secondary bacteremia complicating procedures involving gastrointestinal instrumentation (9).

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Until recently, this organism had not been described except as a hitherto-unknown, Gram-negative, tiny, anaerobic coccus, although several “uncultured bacterium” sequences within GenBank also demonstrated a high level of identity with the three recently published Negativicoccus type strain sequences and the sequence from the blood isolate (8). All of the three recently characterized clinical isolates were recovered from skin and soft tissue samples. In the cases of these three isolates, nearly complete 16S rRNA gene sequences (1,401 nucleotides [nt]) and partial dnaK sequences (about 650 nt) were matched with sequences in the GenBank and EMBL databases using the BLAST program (http://blast.ncbi.nlm.nih.gov). Our blood isolate also demonstrated biochemical reactions similar to those of the three skin and soft tissue isolates previously described, although we did not test for metabolic end products (8). The cells of these isolates were tiny (0.4 ␮m in diameter), coccoid, Gram negative, nonmotile, and nonsporulating. Colonies on Columbia blood agar are very tiny (less than 0.5 mm in diameter after 48 h), circular, convex, and translucent. The organism grows best under anaerobic conditions, but it is asaccharolytic (fructose, glucose, lactose, maltose, mannose, and sucrose) and unreactive in common conventional biochemical tests (nitrate reduction, nitrite reduction, gas production, catalase, indole, and urease). Growth may be enhanced in the presence of sodium succinate (8). Our blood isolate and the three previously described skin isolates displayed susceptibility to bile (1-␮g disk) and were sensitive to kanamycin (500 ␮g) but resistant to vancomycin (5-␮g disk) and colistin (10-␮g disk). The three skin isolates also had arginine arylamidase and alkaline phosphatase activity, whereas our isolate had only PyrA activity by Vitek ANC card analysis. The metabolic end products produced by the three skin isolates were acetic, propionic, and lactic acids, as well as 2-hydroxyvaleric acid in trace amounts (8). Limited information is available regarding the putative antimicrobial susceptibility profiles of Negativicoccus. A major difference between our blood isolate and those obtained from clinical skin and soft tissue samples was the susceptibility to metronidazole (8). Our isolate demonstrated a high level of resistance to metronidazole using both the Etest and disk (5 ␮g) diffusion methods, while the three skin and soft tissue strains were susceptible based only on disk testing. However, since this is only the fourth isolate of Negativicoccus reported from human clinical specimens, this difference could also represent the spectrum of antibiotic susceptibility to this compound, and many more isolates will need to undergo antibiotic susceptibility testing to establish this new genus’s typical antibiogram profile.

J. CLIN. MICROBIOL.

In summary, clinical microbiology laboratories should be aware that a new genus, Negativicoccus, has been described within the family Veillonellaceae and that this organism is a normal part of the human skin microbiome (3, 4, 8). However, Negativicoccus also may be part of the normal intestinal flora, as the source of bacteremia in our case. Unusual anaerobic Gram-negative cocci isolated from sterile sites such as blood should be fully identified. Isolates meeting the preliminary phenotypic characteristics of Negativicoccus should be referred for definitive identification using 16S rRNA gene sequencing. Delineation of the clinical significance and pathogenic potential of Negativicoccus in humans is dependent upon further isolation from clinical samples and full phenotypic and genotypic characterization. Nucleotide sequence accession number. The nucleotide sequence of our clinical bacteremia isolate was deposited in GenBank under accession number HQ264056. REFERENCES 1. Clinical and Laboratory Standards Institute. 2008. Interpretive criteria for identification of bacteria and fungi by DNA target sequencing, vol. 12. Approved standard MM18-A. Clinical and Laboratory Standards Institute, Wayne, PA. 2. Edmiston, C. E., Jr., G. R. Avant, and F. A. Wilson. 1982. Anaerobic bacterial populations on normal and diseased human biopsy tissue obtained at colonoscopy. Appl. Environ. Microbiol. 43:1173–1181. 3. Grice, E. A., et al. 2009. Topographical and temporal diversity of the human skin microbiome. Science 324:1190–1193. 4. Grice, E. A., et al. 2008. A diversity profile of the human skin microbiota. Genome Res. 18:1043–1050. 5. Hayashi, H., R. Takahashi, T. Nishi, M. Sakamoto, and Y. Benno. 2005. Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism. J. Med. Microbiol. 54:1093–1101. 6. Hentges, D. J. 1989. Anaerobes as normal flora, p. 37–53. In S. M. Finegold and W. L. George (ed.), Anaerobic infections of humans. Academic Press, Inc., San Diego, CA. 7. Jumas-Bilak, E., et al. 2005. Dialister micraerophilus sp. nov. and Dialister propionicifaciens sp. nov., isolated from human clinical samples. Int. J. Syst. Evol. Microbiol. 55:2471–2478. 8. Marchandin, H., et al. 2010. Negativicoccus succinicivorans gen. nov., sp. nov., isolated from human clinical samples, emended description of the family Veillonellaceae and description of Negativicutes classis nov., Selenomonadales ord. nov. and Acidaminococcaceae fam. nov. in the bacterial phylum Firmicutes. Int. J. Syst. Evol. Microbiol. 60:1271–1279. 9. Marriott, D., D. Stark, and J. Harkness. 2007. Veillonella parvula discitis and secondary bacteremia: a rare infection complicating endoscopy and colonoscopy? J. Clin. Microbiol. 45:672–674. 10. Morotomi, M., F. Nagai, S. Hiroshi, and R. Tanaka. 2008. Dialister succinatiphilus sp. nov. and Barnesiella intestinihominis sp. nov., isolated from human faeces. Int. J. Syst. Evol. Microbiol. 58:2716–2720. 11. Simmon, K. E., A. C. Croft, and C. A. Petti. 2006. Application of SmartGene IDNS software to partial 16S rRNA gene sequences for a diverse group of bacteria in the clinical laboratory. J. Clin. Microbiol. 44:4400–4406. 12. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24:1596–1599.