Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci.
ABSTRACT: To compare commonly used phenotypic methods with genotypic identification methods 47 clinical isolates of coagulase-negative staphylococci (CONS), 10 CONS ATCC strains, and a Staphylococcus aureus clinical isolate were identified using the API Staph ID test, BD Phoenix Automated Microbiology System, and 16S rRNA gene and tuf gene sequencing. When necessary part of the sodA gene was sequenced for definitive identification. The results show that tuf gene sequencing is the best method for identification of CONS, but the API Staph ID test is a reasonably reliable phenotypic alternative. The performance of the BD Phoenix Automated Microbiology System for identification of CONS is poor. The present study also showed that although genotypic methods are clearly superior to phenotypic identifications, a drawback of sequence-based genotypic methods may be a lack of quality of deposited sequences in data banks. In particular, 16S rRNA gene sequencing suffers from the lack of high quality among sequences deposited in GenBank. Furthermore, genotypic identification based on 16S rRNA sequences has limited discriminating power for closely related Staphylococcus species. We propose partial sequencing of the tuf gene as a reliable and reproducible method for identification of CONS species.
Project description:Coagulase-negative staphylococci (CNS) play a predominant role in nosocomial infections. Rapid, reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment of these infections. Quite recently, the VITEK 2 g-positive (gram-positive [GP]) identification card (bioMérieux) has been redesigned for greater accuracy in the identification of gram-positive cocci. We compared the BD Phoenix (Becton Dickinson) and VITEK 2 (bioMérieux) automated microbiology systems, using their respective update version cards, and the API ID32 STAPH test. The glyceraldehyde-3-phosphate dehydrogenase (gap) gene-based T-RFLP (terminal restriction fragment length polymorphism) method was used for verifying the results. In total, 86 clinical isolates of CNS and 27 reference strains were analyzed. The results show that for identification of CNS, the automated identification methods using the newest VITEK 2 and BD Phoenix identification cards are comparable. However, API ID32 STAPH revealed more correct results compared to both automated microbiology systems. Despite the increased performance of the phenotypic automated identification systems compared to the former versions, molecular methods, e.g., the gap-based T-RFLP method, still show superior accuracy in identifying Staphylococcus species other than Staphylococcus aureus.
Project description:Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens from cows with intramammary infection (IMI). Although API STAPH ID 20, a commercially available identification system, and PCR-restriction fragment length polymorphism (PCR-RFLP) of the gap gene (gap PCR-RFLP) have been successfully applied for the identification of CNS isolates from human specimens, their accuracy in the identification of veterinary isolates has not been fully established. In this study, we identified 263 CNS isolates from bovine IMI at species level by partial 16S rRNA gene sequence analysis as the definitive test. Species identification obtained using partial 16S rRNA gene sequence analysis was compared to results from the API STAPH ID 20 and gap PCR-RFLP analysis. Eleven different CNS species were identified by partial 16S rRNA gene sequence analysis. Only 76.0% (200/263) of the species identification results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis, whereas 97.0% (255/263) of the species identification results obtained by the gap PCR-RFLP analysis matched those obtained by partial 16S rRNA gene sequence analysis. The gap PCR-RFLP analysis could be a useful and reliable alternative method for the species identification of CNS isolates from bovine IMI and appears to be a more accurate method of species identification than the API STAPH ID 20 system.
Project description:A real-time PCR assay was developed to identify common staphylococcal species. A single set of consensus primers was designed to amplify a portion of the 16S rRNA gene, and a pair of fluorescence resonance energy transfer probes was used to identify species based on the unique melt properties of the probes resulting from sequence variations in the amplicons from each species. Nine common staphylococcal strains (S. aureus, S. capitis, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, S. schleiferi, S. simulans, and S. warneri) were used for assay development. The species-specific melting profiles were validated by correctly identifying 36 of 37 coagulase-negative staphylococcal (CoNS) isolates identified by ribotyping. In a study of clinical isolates, the PCR/melt curve approach correctly identified 56/56 S. aureus isolates identified by coagulase/protein A latex agglutination. Fifty-four CoNS clinical isolates characterized using the API Staph assay were studied, with the PCR/melt curve approach yielding matching identifications for 32/54 (59%). The API Staph assay was unable to identify 18 CoNS isolates, and differing results were obtained for 4 isolates. Sequencing of the 22 discrepant or unidentified CoNS samples revealed that the PCR/melt curve results were correct for all but one isolate. Thus, PCR/melt curve analysis achieved a nearly 100% accuracy and performed better than biochemical testing. Performance of the PCR/melt curve approach requires less than 2 h after colony selection. This method thus provides a rapid and accurate approach to the identification of staphylococcal species in the clinical laboratory.
Project description:The terminology and classification of the Anginosus group streptococci has been inconsistent. We tested the utility of 16S rRNA gene and tuf gene sequencing and conventional biochemical tests for the reliable differentiation of the Anginosus group streptococci. Biochemical testing included Rapid ID 32 Strep, API Strep, Fluo-Card Milleri, Wee-tabs, and Lancefield antigen typing. Altogether, 61 Anginosus group isolates from skin and soft tissue infections and four reference strains were included. Our results showed a good agreement between 16S rRNA gene and tuf gene sequencing. Using the full sequence was less discriminatory than using the first part of the 16S rRNA gene. The three species could not be separated with the API 20 Strep test. Streptococcus intermedius could be differentiated from the other two species by beta-galactosidase (ONPG) and beta-N-acetyl-glucosaminidase reactions. Rapid ID 32 Strep beta-glucosidase reaction was useful in separating S. anginosus strains from S. constellatus. In conclusion, both 16S rRNA gene and tuf gene sequencing can be used for the reliable identification of the Anginosus group streptococci. S. intermedius can be readily differentiated from the other two species by phenotypic tests; however, 16S rRNA gene or tuf gene sequencing may be needed for separating some strains of S. constellatus from S. anginosus.
Project description:BACKGROUND: The staphylococci are one of the most common environmental isolates found in clean room facility. Consequently, isolation followed by comprehensive and accurate identification is an essential step in any environmental monitoring program. FINDINGS: We have used the API Staph identification kit (bioMérieux, France) which depends on the expression of metabolic activities and or morphological features to identify the Staphylococcus isolates. The API staphylococci showed low sensitivity in the identification of some species, so we performed molecular methods based on PCR based fingerprinting of glyceraldehyde-3-phosphate dehydrogenase encoding gene as useful taxonomic tool for examining Staphylococcus isolates. CONCLUSIONS: Our results showed that PCR protocol used in this study which depends on genotypic features was relatively accurate, rapid, sensitive and superior in the identification of at least 7 species of Staphylococcus than API Staph which depends on phenotypic features.
Project description:Coagulase-negative staphylococcus isolates were identified using Sensititre GPID plates and API strips (n = 156). For selected isolates, partial sequencing of the 16S rRNA, sodA, and tuf genes was performed. The Sensititre plates correctly identified 68.9% of isolates, with a concordance of 86% for Staphylococcus haemolyticus and 73% for Staphylococcus epidermidis.
Project description:AIMS:To ascertain the clinical importance of a strain of slide coagulase positive but tube coagulase negative Staphylococcus species isolated from the blood culture of a 43 year old patient with refractory anaemia with excessive blasts in transformation who had neutropenic fever. METHODS:The isolate was investigated phenotypically by standard biochemical methods using conventional biochemical tests and two commercially available systems, the Vitek (GPI) and API (Staph) systems. Genotypically, the 16S ribosomal RNA (rRNA) gene of the bacteria was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank by multiple sequence alignment. RESULTS:Conventional biochemical tests did not reveal a pattern resembling a known Staphylococcus species. The Vitek system (GPI) showed that it was 94% S. simulans and 3% S. haemolyticus, whereas the API system (Staph) showed that it was 86.8% S. aureus and 5.1% S. warneri. 16S rRNA gene sequencing showed that there was a 0 base difference between the isolate and S. aureus, 28 base difference between the isolate and S. lugdunensis, 39 base difference between the isolate and S. schleiferi, 21 base difference between the isolate and S. haemolyticus, 41 base difference between the isolate and S. simulans, and 23 base difference between the isolate and S. warneri, indicating that the isolate was a strain of S. aureus. Vancomycin was subsequently prescribed and blood cultures taken four days after the start of treatment were negative. CONCLUSIONS:16S rRNA gene sequencing was useful in ascertaining the clinical importance of the strain of slide coagulase positive but tube coagulase negative Staphylococcus species isolated from blood culture and allowing appropriate management.
Project description:The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria. Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data, the proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and microheterogeneity in 16S rRNA gene sequence within a species is common. Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.
Project description:Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported.We designed a new set of primers and amplified a 420-444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter-/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases.This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.
Project description:The diagnosis of bloodstream infection with coagulase-negative staphylococci is frequently based on the isolation of the same organism from more than one blood culture. Phenotypic variation is a common characteristic of pathogenic strains of Staphylococcus epidermidis which may affect species identification by the microbiology laboratory. We describe a patient with a new onset of nephritis and gram-positive bacteremia. Gram-positive cocci grew in multiple blood cultures and were identified by the Vitek 2 system as Kocuria varians, Staphylococcus hyicus, and S. epidermidis. Bacterial isolates grew on blood agar and Congo red agar plates as two distinct morphotypes and exhibited phenotypic variation. Neither morphotype could be identified by the API-Staph assay. Cellular fatty acid analysis identified one of the morphotypes as S. epidermidis but could not identify the other morphotype. All isolates were found to be identical by pulsed-field gel electrophoresis, and both colonial morphotypes were identified as S. epidermidis by 16S ribosomal gene sequencing. Phenotypic variation of S. epidermidis may affect identification to the species level by phenotype-based identification systems. Caution should be exercised when differentiating between true infection and contamination based on strain identification.