Manual and Automated Instrumentation for Identification of<i>Enterobacteriaceae</i>and Other Aerobic Gram-Negative Bacilli

O'Hara, C M

doi:10.1128/cmr.18.1.147-162.2005

Cited by 116 publications

(78 citation statements)

References 85 publications

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“…To solve this problem, investigators [4,17,20,25,27,[29][30][31] sought universal primers. Unfortunately, ''Universal Primers'' do not live up to their name since they do not cover all bacteria [30].…”

Section: Introductionmentioning

confidence: 99%

A Universal Method for the Identification of Bacteria Based on General PCR Primers

Barghouthi

2011

Indian J Microbiol

102

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The Universal Method (UM) described here will allow the detection of any bacterial rDNA leading to the identification of that bacterium. The method should allow prompt and accurate identification of bacteria. The principle of the method is simple; when a pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial DNA data base, then the bacterium can be identified. Confirmation of identity may follow. In this work, several general 16S primers were designed, mixed and applied successfully against 101 different bacterial isolates. One mixture, the Golden mixture7 (G7) detected all tested isolates (67/67). Other golden mixtures; G11, G10, G12, and G5 were useful as well. The overall sensitivity of the UM was 100% since all 101 isolates were detected yielding intended PCR amplicons. A selected PCR band from each of 40 isolates was sequenced and the bacterium identified to species or genus level using BLAST. The results of the UM were consistent with bacterial identities as validated with other identification methods; cultural, API 20E, API 20NE, or genera and species specific PCR primers. Bacteria identified in the study, covered 34 species distributed among 24 genera. The UM should allow the identification of species, genus, novel species or genera, variations within species, and detection of bacterial DNA in otherwise sterile samples such as blood, cerebrospinal fluid, manufactured products, medical supplies, cosmetics, and other samples. Applicability of the method to identifying members of bacterial communities is discussed. The approach itself can be applied to other taxa such as protists and nematodes.

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Section: Introductionmentioning

confidence: 99%

A Universal Method for the Identification of Bacteria Based on General PCR Primers

Barghouthi

2011

Indian J Microbiol

102

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“…In a routine clinical laboratory, species identification of cultured isolates usually relies on phenotypic methods, such as panels of biochemical reactions, antibiotic resistance, and fatty acid patterns (28). However, due to their limited biochemical reactivity and variable morphology, nonfermenters are frequently misidentified by classical methods (21,26).…”

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Evaluation of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry in Comparison to 16S rRNA Gene Sequencing for Species Identification of Nonfermenting Bacteria

et al. 2008

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Nonfermenting bacteria are ubiquitous environmental opportunists that cause infections in humans, especially compromised patients. Due to their limited biochemical reactivity and different morphotypes, misidentification by classical phenotypic means occurs frequently. Therefore, we evaluated the use of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for species identification. By using 248 nonfermenting culture collection strains composed of 37 genera most relevant to human infections, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurement and MALDI BioTyper software (Bruker Daltonik GmbH, Leipzig, Germany), i.e., by using a mass range of 2,000 to 20,000 Da and a new pattern-matching algorithm. To evaluate the database, 80 blind-coded clinical nonfermenting bacterial strains were analyzed. As a reference method for species designation, partial 16S rRNA gene sequencing was applied. By 16S rRNA gene sequencing, 57 of the 80 isolates produced a unique species identification (>99% sequence similarity); 11 further isolates gave ambiguous results at this threshold and were rated as identified to the genus level only. Ten isolates were identified to the genus level (>97% similarity); and two isolates had similarity values below this threshold, were counted as not identified, and were excluded from further analysis. MALDI-TOF MS identified 67 of the 78 isolates (85.9%) included, in agreement with the results of the reference method; 9 were misidentified and 2 were unidentified. The identities of 10 randomly selected strains were 100% correct when three different mass spectrometers and four different cultivation media were used. Thus, MALDI-TOF MS-based species identification of nonfermenting bacteria provided accurate and reproducible results within 10 min without any substantial costs for consumables.

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“…The performance of some automated systems has been evaluated with regard to catalase-negative, gram-positive cocci (7,13,14,17,24). Reproducibility and accuracy of results, turnaround time, availability of data for epidemiological monitoring, and cost-effectiveness constitute the main reasons supporting the choice of automated systems.Becton Dickinson (BD Diagnostic Systems, Sparks, MD) has introduced the Phoenix automated microbiology system for ID and antimicrobial susceptibility testing (AST) of human pathogenic bacteria, including enterobacteria, nonfermenting gram-negative bacteria, staphylococci, and enterococci (5,8,11,25). Recently, the SMIC/ID-2 panel, dedicated to ID and AST of streptococcal species, was launched (15, 18).…”

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confidence: 99%

“…Becton Dickinson (BD Diagnostic Systems, Sparks, MD) has introduced the Phoenix automated microbiology system for ID and antimicrobial susceptibility testing (AST) of human pathogenic bacteria, including enterobacteria, nonfermenting gram-negative bacteria, staphylococci, and enterococci (5,8,11,25). Recently, the SMIC/ID-2 panel, dedicated to ID and AST of streptococcal species, was launched (15, 18).…”

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confidence: 99%

Use of the Phoenix Automated System for Identification of Streptococcus and Enterococcus spp

et al. 2006

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The Phoenix system (Becton Dickinson Diagnostic Systems, Sparks, MD) was evaluated for identification (ID) to the species level of streptococci and enterococci. Two hundred clinical isolates were investigated: beta-hemolytic streptococci (n ‫؍‬ 50), Streptococcus pneumoniae organisms (n ‫؍‬ 46), viridans group streptococci (n ‫؍‬ 31), Enterococcus faecium (n ‫؍‬ 36), Enterococcus faecalis (n ‫؍‬ 25), and other catalase-negative cocci (n ‫؍‬ 12). The API system (bioMérieux, Marcy l'Étoile, France) was used as a comparator. Molecular methods (sequencing of 16S rRNA and zwf and gki genes and ddl gene amplification) were used to investigate discordant results. Upon resolution of discrepancies, correct species ID was achieved by the Phoenix system for 121/129 (93.8%) streptococci and 63/70 (90.0%) enterococci. Excellent results were obtained for S. pneumoniae (45/45) and beta-hemolytic streptococci (49/50). With regard to viridans streptococci, the accuracy of the Phoenix system was 83.9%. Among the latter organisms, the best performance was obtained with isolates of the Streptococcus sanguinis group and Streptococcus anginosus group; problems were instead encountered with the Streptococcus mitis group. Four E. faecium and three E. faecalis isolates were misidentified as Enterococcus casseliflavus/Enterococcus gallinarum or Enterococcus durans. Thus, these isolates were identified only at the genus level. Compared with commercially available systems, the Phoenix system appears a reliable diagnostic tool for identifying clinically relevant streptococci and enterococci. The SMIC/ID-2 panel proved particularly effective for beta-hemolytic streptococci and pneumococci.Catalase-negative, gram-positive cocci are a heterogeneous group of 17 genera that include streptococci, enterococci, and nonstreptococcal, nonenterococcal species (9, 10). Over 70 streptococcal and enterococcal species have been implicated in human disease (10,27). Of these, only a few are known to cause important infections (e.g., Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, and viridans group streptococci).A number of manual, semiautomated, and automated systems are reported to produce acceptable identification (ID) results for S. pneumoniae, beta-hemolytic streptococci, and enterococcal species (26,27). These systems, however, were shown not to be sufficiently accurate in identifying streptococci of the viridans group (13, 20), organisms of complex taxonomy (2, 10, 26). The performance of some automated systems has been evaluated with regard to catalase-negative, gram-positive cocci (7,13,14,17,24). Reproducibility and accuracy of results, turnaround time, availability of data for epidemiological monitoring, and cost-effectiveness constitute the main reasons supporting the choice of automated systems.Becton Dickinson (BD Diagnostic Systems, Sparks, MD) has introduced the Phoenix automated microbiology system for ID and antimicrobial susceptibility testing (AST) of human pathogen...

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Manual and Automated Instrumentation for Identification ofEnterobacteriaceaeand Other Aerobic Gram-Negative Bacilli

Cited by 116 publications

References 85 publications

A Universal Method for the Identification of Bacteria Based on General PCR Primers

A Universal Method for the Identification of Bacteria Based on General PCR Primers

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry in Comparison to 16S rRNA Gene Sequencing for Species Identification of Nonfermenting Bacteria

Use of the Phoenix Automated System for Identification of Streptococcus and Enterococcus spp

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