Abstract
Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non‐duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system. The MicroSeq system successfully identified 13 of the 20 isolates. Four and three isolates were misidentified at the genus and species level, respectively. Although the MicroSeq 500 16S rDNA bacterial identification system is better than three commercially available identification systems also evaluated, its database needs to be expanded for accurate identification of anaerobic Gram positive bacilli.
Keywords: Microseq 500, 16S rDNA gene, anaerobic Gram positive bacilli, blood cultures
Identification of anaerobic Gram positive bacilli in clinical microbiology laboratories by phenotypic methods is often difficult. Comparison of the gene sequences of bacterial species has shown that the 16S ribosomal RNA (rRNA) gene is highly conserved within a species and among species of the same genus. Hence, it can be used as the new standard for classification and identification of bacteria.1,2 Recently, we reported the application of this technique for identifying this group of bacteria.3,4,5,6,7 The MicroSeq 500 16S rDNA bacterial identification system (Perkin‐Elmer Applied Biosystems Division, Foster City, California, USA) has been designed for rapid and accurate identification of bacterial pathogens, using the first 527 bp fragment of the 16S rRNA gene. It has been shown that the system is useful for the identification of unusual aerobic pathogenic Gram negative bacilli, coryneform bacteria, mycobacterium, and nocardia species, and various bacterial strains with ambiguous biochemical profiles.8,9,10,11,12 In our study, we evaluate the usefulness of this system in the identification of 20 non‐duplicating anaerobic Gram positive bacilli isolated from blood cultures.
“The MicroSeq 500 16S rDNA bacterial identification system is useful for the identification of unusual aerobic pathogenic Gram negative bacilli, coryneform bacteria, mycobacterium, and nocardia species, and various bacterial strains with ambiguous biochemical profiles”
Materials and methods
Bacterial strains
The bacterial strains were isolates from blood cultures of patients hospitalised at the Queen Mary Hospital in Hong Kong during a four year period (January 1998 to December 2001). Isolates were identified as Clostridium perfringens and Propionibacterium acnes by phenotypic methods. One isolate each of C perfringens and P acnes and all isolates other than C perfringens and P acne were subjected to 16S rRNA gene sequencing. One isolate for each species was selected for DNA sequencing of the first 527 bp fragment of the 16S rRNA gene and analysis by the MicroSeq 16S rDNA bacterial identification system, in addition to identification by three commercially available identification systems for anaerobes: the Vitek System (ANI; bioMerieux Vitek, USA, Hazelwood, Missouri, USA), the RapID ANA II system (Innovative Diagnostic Systems, Atlanta, Georgia, USA), and the API system (20A; bioMerieux Vitek). Each isolate was categorised as clinically significant or a contaminant (pseudobacteraemia) by criteria described previously.13
Conventional 16S rRNA gene sequencing
Polymerase chain reaction amplification and DNA sequencing of the full 16S rRNA genes were performed according to our previous publications.3,7,14 Strains 1–13, 15–17, and 20 were amplified with primers LPW58 (5′‐AGGCCCGGGAACGTATTCAC‐3′) and LPW81 (5′‐TGGCGAACGGGTGAGTAA‐3′), strains 14 and 19 with primers LPW55 (5′‐AGTTTGATCCTGGCTCAG‐3′) and LPW325 (5′‐CGGATACCTTGTTACGACT‐3′), and strain 18 with primers LPW55 (5′‐AGTTTGATCCTGGCTCAG‐3′) and LPW205 (5′‐CTTGTTACGACTTCACCC‐3′). The sequences of the polymerase chain reaction products were compared with known 16S rRNA gene sequences in the GenBank by multiple sequence alignment using the CLUSTAL W program.15
Identification by the MicroSeq 500 16S rDNA bacterial identification system
Bacterial DNA extracts were amplified with 0.5 μM primers (005F and 531R) according to the manufacturer's instructions. The DNA sequences were analysed using the database provided by the system.
Results
Patient characteristics
Twenty strains, representing 20 non‐duplicating anaerobic Gram positive bacilli, were selected for further analysis by the MicroSeq 16S rDNA bacterial identification system and identification by three commercially available identification systems. Table 1 summarises the characteristics of the 20 patients. The clinical details of patients 14 and 18 have been described previously.3,7
Table 1 Identification of anaerobic Gram positive bacterial isolates by conventional 16S rRNA gene sequencing, commercially available bacterial identification systems, and the Microseq 500 16S rDNA bacterial identification system.
Patient/strain no. | Patient characteristics | Conventional 16S rRNA gene sequencing | Commercially available bacterial identification systems | MicroSeq 500 16S rDNA bacterial identification system | ||||
---|---|---|---|---|---|---|---|---|
Sex/age* | Diagnosis | Vitek ANI | RapID ANA II | API 20A | Identity | % Difference between isolate sequences and closest match | ||
1 | M/1 m | Pseudobacteraemia | Bifidobacterium pseudocatenulatum/catenulatum | Unidentified | >99.9% Clostridium perfringens | 92% Eubacterium lentum, 8% Actinomyces viscosus | B catenulatum | 1.4 |
2 | F/94 | Pseudobacteraemia | Clostridium barati | Unidentified | 99.5% C barati | 68% Actinomyces israelii, 17% Bifidobacterium sp. | C barati | 0 |
3 | M/1 m | Necrotising enterocolitis | Clostridium difficile | 94% C difficile | >99.9% C difficile | 99.9% C difficile | C difficile | 0 |
4 | F/75 | Primary bacteraemia | Clostridium disporicum | 99% C barati | >99.9% C barati | 94% E lentum, 3% A viscosus | Clostridium paraputrificum | 3.3 |
5 | F/77 | Pseudobacteraemia | Clostridium indolis | 89% Clostridium tetani | 73% Clostridium limosum, 16% Clostridium novyi A, 12% C tetani | Unidentified | Clostridium innocuum | 8.7 |
6 | M/80 | Pseudobacteraemia | C innocuum | 61% Corynebacterium jeikeium, 37% Lactobacillus jensenii | 74% C innocuum, 26% Clostridium subterminale | 98% C innocuum | Eubacterium dolichum | 8.0 |
7 | F/24 | Pseudobacteraemia | Clostridium orbiscindens | 45% C subterminale, 39% Clostriduim histolyticum, 11% C jeikeium | 67% C tetani, 19% novyi A, 12% E lentum | 94% E lentum, 3% A viscosus | Ruminococcus productus | 18.4 |
8 | M/6 m | Intussusception | C paraputrificum | 81% Clostridium septicum | 77% C septicum, 23% C paraputrificum | 80% C paraputrificum, 19% C barati | C paraputrificum | 0 |
9 | F/44 | Acute cholangitis | Clostridium perfringens | 99% C perfringens | >99.9% C perfringens | 97% C perfringens, 3% Actinomyces naeslundii | C perfringens | 0.2 |
10 | M/54 | Primary bacteraemia | Clostridium ramosum | 83% C barati, 11% Lactobacillus catenaforme | >99.9% C ramosum | 96% C ramosum, 3% Bifidobacterium sp. | C ramosum | 0 |
11 | F/40 | Neutropenic fever | C septicum | 54% C septicum, 42% C paraputrificum | >99.9% C septicum | 99.9% C septicum | Clostridium tertium | 2.6 |
12 | F/45 | Pseudobacteraemia | Clostridium sporosphaeroides | Unidentified | 67% C tetani, 19% C novyi A, 12% E lentum | Unidentified | C sporosphaeroides | 10.6 |
13 | F/78 | Pseudobacteraemia | C tertium | 71% C tertium, 22% Clostridium clostridiforme | 99.9% C barati | 90% C tertium, 7% Bifidobacterium sp. | C tertium | 0 |
14 | F/87 | Infected bed sore | Eggerthella lenta | 83% C jeikeium, 7% Clostridium hastiforme, 7% C histolyticum | >99.9% E lenta | 92% E lenta, 8% A viscosus | E lenta | 0 |
15 | F/85 | Primary bacteraemia | Eubacterium tenue | 81% Propionibacterium granulosum, 13% Actinomyces odontolyticus | 96% Clostridium sordellii, 4% Clostridium bifermentans | 97% A viscosus, 2% E lentum | Clostridium tenue | 1.4 |
16 | F/41 | Primary bacteraemia | Lactobacillus casei/paracasei | Unidentified | Unidentified | 97% Bifidobacterium sp., 3% A israelii | L casei/paracasei | 0 |
17 | M/50 | Primary bacteraemia | Lactobacillus rhamnosus | 50% Lactobacillus jensenii, 46% Actinomyces odontolyticus | >99.9% Lactobacillus acidophilus | 63% A naeslundii, 27% Lactobacillus acidophilus/jensenii | L rhamnosus | 0 |
18 | M/70 | Acute cholecystitis | Lactobacillus salivarius | Unidentified | >99.9% Propionibacterium granulosum | 70% A naeslundii, 30% Bifidobacterium sp. | L salivarius | 0.1 |
19 | M/43 | Acute cholangitis | Olsenella uli | 81% P granulosum, 11% Corynebacterium pseudotuberculosis | Unidentified | 43% Gemella morbillorum, 37% Lactobacillus fermentum, 10% Propionicum/avidum | Atopobium rimae | 11.1 |
20 | F/1 m | Pseudobacteraemia | Propionibacterium acnes | 99.9% P acnes | >99.9% P acnes | 99.9% P acnes | P acnes | 0 |
*In years or months (m).
Conventional 16S ribosomal RNA gene sequencing
Table 1 shows the results of 16S rRNA gene sequence analysis. For all the 20 isolates, there was < 2% difference between the 16S rRNA gene sequences of the isolates and the most closely matched sequence in the GenBank.
Identification by the MicroSeq 500 16S rDNA bacterial identification system
The identities of 13 strains were consistent with those obtained by conventional 16S rRNA gene sequencing (table 1). For the remaining seven sequences, four isolates were misidentified at the genus level (strain 6, C innocuum misidentified as Eubacterium dolichum; strain 7, C orbiscindens misidentified as Ruminococcus productus; strain 15, E tenue misidentified as C tenue; and strain 19, Olsenella uli misidentified as Atopobium rimae), whereas three were misidentified at the species level (strain 4, C disporicum misidentified as C paraputrificum; strain 5, C indolis misidentified as C innocuum; and strain 11, C septicum misidentified as C tertium).
Identification by commercially available bacterial identification systems
The Vitek ANI system was able to identify 10 and four of the 20 isolates, the RapID ANA II system 15 and eight isolates, and the API 20A system nine and nine isolates to the genus and species levels with > 70% confidence, respectively (table 1).
Discussion
Although the MicroSeq 500 16S rDNA bacterial identification system was better than the three commercially available systems in the identification of the 20 anaerobic Gram positive bacilli tested in our present study, its accuracy is still suboptimal. Using conventional 16S rRNA gene sequencing as the gold standard, the MicroSeq 500 16S rRNA bacterial identification system was able to identify 16 of the 20 (80%) isolates to the genus level, and only 13 (65%) of the isolates to the species level in our present study, compared with the corresponding figures of 86.5% and 81.1% in our previous study on bacterial strains of more diverse genera and species,12 and 97.2% and 89.2% in a study on unusual aerobic Gram negative bacilli.11
Take home messages
The MicroSeq 500 16S rDNA bacterial identification system identified 13 of 20 non‐duplicating anaerobic Gram positive bacilli isolated from blood cultures
The system compared favourably with three other commercially available identification systems also evaluated
However, the system's database needs to be expanded for accurate identification of anaerobic Gram positive bacilli
“The database of the MicroSeq 500 16S rDNA bacterial identification system needs to be expanded to improve its accuracy in the identification of anaerobic Gram positive bacilli”
The most common reason for the MicroSeq 500 16S rDNA bacterial identification system to fail to identify a bacterium was a lack of the 16S rRNA gene sequence of the particular bacterium in the database, which is in line with results from our previous study.12 The 16S rRNA gene sequences of five of the misidentified isolates were not included in the system database, probably because they are rarely encountered. When the same 527 bp DNA sequences of these seven misidentified isolates were compared with the known 16S rRNA gene sequences in the GenBank, six yielded the correct identity, with good discrimination between the best and second best match sequences (table 2). Thus, the database of the MicroSeq 500 16S rDNA bacterial identification system needs to be expanded to improve its accuracy in the identification of anaerobic Gram positive bacilli.
Table 2 Analysis of DNA sequences of strains identified incorrectly using the Microseq 500 16S rDNA bacterial identification system database.
Patient/strain no. | Identification by conventional 16S rRNA gene sequencing | Identification by DNA sequencing of first 527 bp fragment of 16S rRNA gene | ||||
---|---|---|---|---|---|---|
Using MicroSeq 500 16S rDNA database | Analysis using the GenBank database | |||||
BM | No. of base (%) difference between strain and BM | 2nd BM | No. of base (%) difference between strain and 2nd BM | |||
4 | Clostridium disporicum | Clostridium paraputrificum | C disporicum | 10 (2.1) | Clostridium gasigenes | 24 (4.8) |
5 | Clostridium indolis | Clostridium innocuum | C indolis | 14 (2.7) | Clostridium symbiosum | 45 (8.7) |
6 | C innocuum | Eubacterium dolichum | C innocuum | 9 (1.7) | Eubacterium cylindroides | 17 (5.4) |
7 | Clostridium orbiscindens | Ruminococcus productus | C orbiscindens | 3 (0.6) | Bacteroides capillosus | 29 (5.8) |
11 | Clostridium septicum | Clostridium tertium | C septicum | 1 (0.2) | Clostridium chauvoei | 9 (1.8) |
15 | Eubacterium tenue | Clostridium tenue | Clostridium ghonii | 12 (2.4) | Clostridium bifermentans | 15 (3.1) |
19 | Olsenella uli | Atopobium rimae | O uli | 0 (0) | Olsenella profusa | 21 (4.3) |
BM, best match.
Acknowledgements
This work was partly supported by the University Research Grant Council Grant (HKU 7236/02M), and the Committee for Research and Conference Grant, The University of Hong Kong.
Abbreviations
rRNA - ribosomal RNA
References
- 1.Olsen G J, Woese C R. Ribosomal RNA: a key to phylogeny. FASEB J 19937113–123. [DOI] [PubMed] [Google Scholar]
- 2.Relman D A, Loutit J S, Schmidt T M.et al The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. N Engl J Med 19903231573–1580. [DOI] [PubMed] [Google Scholar]
- 3.Woo P C Y, Fung A M Y, Lau S K P.et al Identification by 16S ribosomal RNA gene sequencing of Lactobacillus salivarius bacteremic cholecystitis. J Clin Microbiol 200240265–267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Woo P C Y, Fung A M Y, Lau S K P.et al Diagnosis of pelvic actinomycosis by 16S ribosomal RNA gene sequencing and its clinical significance. Diagn Microbiol Infect Dis 200243113–118. [DOI] [PubMed] [Google Scholar]
- 5.Woo P C Y, Lau S K P, Woo G K S.et al Bacteremia due to Clostridium hathewayi in a patient with acute appendicitis. J Clin Microbiol 2004425947–5949. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Woo P C Y, Fung A M Y, Lau S K P.et al Actinomyces hongkongensis sp. nov. A novel Actinomyces species isolated from a patient with pelvic actinomycosis. Syst Appl Microbiol 200326518–522. [DOI] [PubMed] [Google Scholar]
- 7.Lau S K P, Woo P C Y, Woo G K S.et al Eggerthella hongkongensis sp. nov. and Eggerthella sinensis sp. nov. two novel Eggerthella species, account for half of the cases of Eggerthella bacteremia. Diagn Microbiol Infect Dis 200449255–263. [DOI] [PubMed] [Google Scholar]
- 8.Cloud J L, Conville P S, Croft A.et al Evaluation of partial 16S ribosomal DNA sequencing for identification of nocardia species by using the MicroSeq 500 system with an expanded database. J Clin Microbiol 200442578–584. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Patel J B, Leonard D G, Pan X.et al Sequence‐based identification of Mycobacterium species using the Microseq 500 16S rDNA bacterial identification system. J Clin Microbiol 200038246–251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Tang Y W, Von Graevenitz A, Waddington M G.et al Identification of coryneform bacterial isolates by ribosomal DNA sequence analysis. J Clin Microbiol 2000381676–1678. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Tang Y W, Ellis N M, Hopkins M K.et al Comparison of phenotypic and genotypic technique for identification of unusual aerobic pathogenic gram‐negative bacilli. J Clin Microbiol 1998363674–3679. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Woo P C, Ng K H, Lau S K.et al Usefulness of the MicroSeq 500 16S ribosomal DNA‐based bacterial identification system for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. J Clin Microbiol 2003411996–2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Weinstein M P, Towns M L, Quartey S M.et al The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults. Clin Infect Dis 199724584–602. [DOI] [PubMed] [Google Scholar]
- 14.Lau S K P, Woo P C Y, Tse H.et al Invasive Streptococcus iniae infections outside North America. J Clin Microbiol 2003411004–1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Thompson J D, Higgins D G, Gibson T J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting position‐specific gap penalties and weight matrix choice. Nucleic Acids Res 1994224673–4680. [DOI] [PMC free article] [PubMed] [Google Scholar]