This study is believed to be the first to provide guidelines for facilitating interpretation of results based on full and 527 bp 16S rRNA gene sequencing and MicroSeq databases used for identifying medically important aerobic Gram-positive bacteria. Overall, full and 527 bp 16S rRNA gene sequencing can identify 24 and 40 % of medically important Gram-positive cocci (GPC), and 21 and 34 % of medically important Gram-positive rods (GPR) confidently to the species level, whereas the full-MicroSeq and 500-MicroSeq databases can identify 15 and 34 % of medically important GPC and 14 and 25 % of medically important GPR confidently to the species level. Among staphylococci, streptococci, enterococci, mycobacteria, corynebacteria, nocardia and members of Bacillus and related taxa (Paenibacillus, Brevibacillus, Geobacillus and Virgibacillus), the methods and databases are least useful for identification of staphylococci and nocardia. Only 0–2 and 2–13 % of staphylococci, and 0 and 0–10 % of nocardia, can be confidently and doubtfully identified, respectively. However, these methods and databases are most useful for identification of Bacillus and related taxa, with 36–56 and 11–14 % of Bacillus and related taxa confidently and doubtfully identified, respectively. A total of 15 medically important GPC and 18 medically important GPR that should be confidently identified by full 16S rRNA gene sequencing are not included in the full-MicroSeq database. A total of 9 medically important GPC and 21 medically important GPR that should be confidently identified by 527 bp 16S rRNA gene sequencing are not included in the 500-MicroSeq database. 16S rRNA gene sequence results of Gram-positive bacteria should be interpreted with basic phenotypic tests results. Additional biochemical tests or sequencing of additional gene loci are often required for definitive identification. To improve the usefulness of the MicroSeq databases, bacterial species that can be confidently identified by 16S rRNA gene sequencing but are not found in the MicroSeq databases should be included.
BosshardP. P.,
ZbindenR.,
AbelsS.,
BöddinghausB.,
AltweggM.,
BöttgerE. C.2006; 16S rRNA gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of nonfermenting Gram-negative bacteria in the clinical laboratory. J Clin Microbiol 44:1359–1366[CrossRef]
CloudJ. L.,
ConvilleP. S.,
CroftA.,
HarmsenD.,
WitebskyF. G.,
CarrollK. C.2004; 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 42:578–584[CrossRef]
CloudJ. L.,
HogganK.,
BelousovE.,
CohenS.,
Brown-ElliottB. A.,
MannL.,
WilsonR.,
AldousW.,
WallaceR. J.Jr,
WoodsG. L.2005; Use of the MGB Eclipse system and SmartCycler PCR for differentiation of Mycobacterium chelonae and M. abscessus
. J Clin Microbiol 43:4205–4207[CrossRef]
CLSI2008Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing , approved guideline MM18-A Wayne, PA: Clinical and Laboratory Standards Institute;
ConvilleP. S.,
ZelaznyA. M.,
WitebskyF. G.2006; Analysis of secA1 gene sequences for identification of Nocardia species. J Clin Microbiol 44:2760–2766[CrossRef]
DrancourtM.,
BolletC.,
CarliozA.,
MartelinR.,
GayralJ. P.,
RaoultD.2000; 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol 38:3623–3630
FontanaC.,
FavaroM.,
PelliccioniM.,
PistoiaE. S.,
FavalliC.2005; Use of the MicroSeq 500 16S rRNA gene-based sequencing for identification of bacterial isolates that commercial automated systems failed to identify correctly. J Clin Microbiol 43:615–619[CrossRef]
GauduchonV.,
ChalabreysseL.,
EtienneJ.,
CélardM.,
BenitoY.,
LepidiH.,
Thivolet-BéjuiF.,
VandeneschF.2003; Molecular diagnosis of infective endocarditis by PCR amplification and direct sequencing of DNA from valve tissue. J Clin Microbiol 41:763–766[CrossRef]
HeikensE.,
FleerA.,
PaauwA.,
FlorijnA.,
FluitA. C.2005; Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci. J Clin Microbiol 43:2286–2290[CrossRef]
JandaJ. M.,
AbbottS. L.2007; 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J Clin Microbiol 45:2761–2764[CrossRef]
KotilainenP.,
HeiroM.,
JalavaJ.,
RantakokkoV.,
NikoskelainenJ.,
NikkariS.,
Rantakokko-JalavaK.2006; Aetiological diagnosis of infective endocarditis by direct amplification of rRNA genes from surgically removed valve tissue. An 11-year experience in a Finnish teaching hospital. Ann Med 38:263–273[CrossRef]
KwokA. Y.,
SuS. C.,
ReynoldsR. P.,
BayS. J.,
Av-GayY.,
DovichiN. J.,
ChowA. W.1999; Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus
. Int J Syst Bacteriol 49:1181–1192[CrossRef]
LauS. K.,
NgK. H.,
WooP. C.,
YipK. T.,
FungA. M.,
WooG. K.,
ChanK. M.,
QueT. L.,
YuenK. Y.2006; Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures. J Clin Pathol 59:219–222[CrossRef]
MellmannA.,
CloudJ. L.,
AndreesS.,
BlackwoodK.,
CarrollK. C.,
KabaniA.,
RothA.,
HarmsenD.2003; Evaluation of RIDOM, MicroSeq, and GenBank services in the molecular identification of Nocardia species. Int J Med Microbiol 293:359–370[CrossRef]
MurrayP. R.,
BaronE. J.,
JorgensenJ. H.,
LandryM. L.,
PfallerM. A.2007Manual of Clinical Microbiology , 9th edn. Washington, DC: American Society for Microbiology;
PatelJ. B.,
LeonardD. G.,
PanX.,
MusserJ. M.,
BermanR. E.,
NachamkinI.2000; Sequence-based identification of Mycobacterium species using the MicroSeq 500 16S rDNA bacterial identification system. J Clin Microbiol 38:246–251
StackebrandtE.,
GoebelB. M.1994; Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849[CrossRef]
TangY. W.,
EllisN. M.,
HopkinsM. K.,
SmithD. H.,
DodgeD. E.,
PersingD. H.1998; Comparison of phenotypic and genotypic technique for identification of unusual aerobic pathogenic gram-negative bacilli. J Clin Microbiol 36:3674–3679
ThompsonJ. D.,
HigginsD. G.,
GibsonT. J.1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680[CrossRef]
WooP. C.,
TamD. M.,
LeungK. W.,
LauS. K.,
TengJ. L.,
WongM. K.,
YuenK. Y.2002; Streptococcus sinensis sp. nov., a novel Streptococcus species isolated from a patient with infective endocarditis. J Clin Microbiol 40:805–810[CrossRef]
WooP. C.,
FungA. M.,
LauS. K.,
ChanB. Y.,
ChiuS. K.,
TengJ. L.,
QueT. L.,
YungR. W.,
YuenK. Y.2003a; Granulicatella adiacens and Abiotrophia defectiva bacteraemia characterised by 16S rRNA gene sequencing. J Med Microbiol 52:137–140[CrossRef]
WooP. C.,
LauS. K.,
FungA. M.,
ChiuS. K.,
YungR. W.,
YuenK. Y.2003b; Gemella bacteraemia characterized by 16S ribosomal RNA gene sequencing. J Clin Pathol 56:690–693[CrossRef]
WooP. C.,
NgK. H.,
LauS. K.,
YipK. T.,
FungA. M.,
LeungK. W.,
TamD. M.,
QueT. L.,
YuenK. Y.2003c; 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 41:1996–2001[CrossRef]
WooP. C.,
TengJ. L.,
LauS. K.,
LumP. N.,
LeungK. W.,
WongK. L.,
LiK. W.,
LamK. C.,
YuenK. Y.2003e; Analysis of a viridans group strain reveals a case of bacteremia due to Lancefield group G α -hemolytic Streptococcus dysgalactiae subsp. equisimilis in a patient with pyomyositis and reactive arthritis. J Clin Microbiol 41:613–618[CrossRef]
WooP. C.,
TengJ. L.,
LeungK. W.,
LauS. K.,
TseH.,
WongB. H.,
YuenK. Y.2004b; Streptococcus sinensis may react with Lancefield group F antiserum. J Med Microbiol 53:1083–1088[CrossRef]
WooP. C.,
TseH.,
WongS. S.,
TseC. W.,
FungA. M.,
TamD. M.,
LauS. K.,
YuenK. Y.2005; Life-threatening invasive Helcococcus kunzii infections in intravenous drug users and ermA -mediated erythromycin resistance. J Clin Microbiol 43:6205–6208[CrossRef]
WooP. C.,
ChungL. M.,
TengJ. L.,
TseH.,
PangS. S.,
LauV. Y.,
WongV. W.,
KamK. L.,
LauS. K.,
YuenK. Y.2007; In silico analysis of 16S ribosomal RNA gene sequencing-based methods for identification of medically important anaerobic bacteria. J Clin Pathol 60:576–579
WooP. C.,
LauS. K.,
TengJ. L.,
TseH.,
YuenK. Y.2008a; Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect 14:908–934[CrossRef]