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    1. 1. Development and Validation of 16S rRNA Gene Sequencing for Identification of Gram-Positive Rods in Clinical Microbiology laboratory By Indre Budvytiene SFSU, 2008
    2. 2. Summary <ul><li>Overview of routine laboratory methods </li></ul><ul><ul><ul><ul><li>Phenotypic techniques and automation </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Problems with bacterial identification </li></ul></ul></ul></ul><ul><li>Overview of molecular identification methods </li></ul><ul><ul><ul><ul><li>Advantages </li></ul></ul></ul></ul><ul><ul><ul><ul><li>16S rRNA gene </li></ul></ul></ul></ul><ul><li>16S rRNA gene sequencing </li></ul><ul><ul><ul><ul><li>Goals </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Development </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Validation </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Evaluation in unknown Gram-positive rod identification </li></ul></ul></ul></ul><ul><li>Conclusions </li></ul>
    3. 3. Case study <ul><li>70-year-old man with bacteremia and acute cholecystitis was admitted to the hospital. </li></ul><ul><li>Blood and pus from the gallbladder were sent for bacterial culture. </li></ul><ul><li>A non-sporeforming gram-positive rods were isolated from blood and pus cultures. Bacteria grew better in anaerobic environment then aerobic. </li></ul>
    4. 4. Case study <ul><li>API 20A system showed that it was 80% Actinomyces naeslundii and 20% Bifidobacterium species, whereas the Vitek ANI system and the ATB ID32A Expression system showed that it was “unidentified.” </li></ul><ul><li>Isolate was subjected to molecular identification methods and identified as Lactobacillus salivarius. </li></ul><ul><li>If the bacterium had been identified as Actinomyces, penicillin for 6 months would have been the regimen of choice. However, it was Lactobacillus, and a. 2-week course of antibiotic was sufficient. </li></ul>
    5. 5. Case study <ul><li>Accurate and rapid identification of microorganisms is critical for : </li></ul><ul><ul><ul><li>Diagnosis </li></ul></ul></ul><ul><ul><ul><li>Effective patient treatment </li></ul></ul></ul><ul><ul><ul><li>Reduction of overall cost of medical care </li></ul></ul></ul>
    6. 6. Overview of Routine Laboratory Identification Methods
    7. 7. Routine laboratory methods <ul><li>Gram Stain </li></ul>
    8. 8. Routine laboratory methods ( cont.) <ul><li>Biochemical characteristics </li></ul>Catalase test Indole test Fermentation of sugars and H2S production
    9. 9. Routine laboratory methods (cont.) <ul><li>Commercial identification systems </li></ul><ul><ul><li>API , Rapid ANA </li></ul></ul>API identification system Rapid ANA identification system
    10. 10. Routine laboratory methods (cont.) <ul><ul><li>Commercial identification systems </li></ul></ul><ul><ul><li>Microscan, Vitek </li></ul></ul>Vitek - 2
    11. 11. Problems with phynotypical methods <ul><li>Some bacterial isolates stain poorly or exhibit unique catabolic or growth patterns </li></ul>Growth after 48H Ambiguous Gram stain result
    12. 12. Problems with phonotypical methods <ul><li>Biochemical readings are subject of variation and dependent on individual interpretation and expertise. </li></ul>
    13. 13. Advantage of molecular testing in bacterial identification <ul><li>Stable target </li></ul><ul><li>Objective </li></ul><ul><li>No specific growth </li></ul><ul><li>requirements </li></ul><ul><li>Easier to standartize </li></ul>
    14. 14. Examples of Molecular Assays <ul><li>Polymerase chain reaction (PCR) </li></ul><ul><li>Hybridization assay </li></ul><ul><li>Microarrays </li></ul><ul><li>Southern blotting </li></ul><ul><li>Northern blotting </li></ul><ul><li>FISH </li></ul><ul><li>Sequencing </li></ul>
    15. 15. 16S rRNA gene <ul><li>It exists universally among all bacteria. </li></ul><ul><li>It is large enough to provide distinguishing measurements. </li></ul><ul><li>It is essential for recognizing the 5' end of mRNA and hence positioning it correctly on the ribosome. </li></ul><ul><li>The 16S rRNA sequence has been highly conserved. </li></ul>
    16. 16. 16S rRNA gene (cont.) <ul><li>Indre Budvytiene </li></ul><ul><li>It consists of conserved and variable regions </li></ul>
    17. 17. Research goal <ul><li>To develop and optimize 16S rRNA gene sequencing for routine laboratory testing </li></ul><ul><li>To assess its effectiveness in identification of Gram-positive rods, by comparing to conventional methods </li></ul><ul><ul><li>accuracy </li></ul></ul><ul><ul><li>turn-around-time </li></ul></ul><ul><ul><li>labor and cost effects </li></ul></ul>
    18. 18. <ul><li>16S rRNA Gene Sequencing </li></ul>
    19. 19. 16S rRNA gene sequencing process <ul><ul><ul><ul><ul><li>Bacterial colonies </li></ul></ul></ul></ul></ul>DNA extraction PCR product cleanup PCR Cycle sequencing Sequencing product cleanup Sequence detection Data assembly and analysis
    20. 20. 16S rRNA gene sequencing process <ul><ul><ul><ul><ul><li>Bacterial colonies </li></ul></ul></ul></ul></ul>DNA extraction PCR product cleanup PCR Cycle sequencing Sequencing product cleanup Sequence detection Data assembly and analysis
    21. 21. Bacterial DNA Extraction
    22. 22. Bacterial DNA Extraction <ul><ul><li>Bead beating extraction method </li></ul></ul><ul><ul><li>Heating for 10 min at 95-100ºC </li></ul></ul>No DNA present 2.0 McFarland solution
    23. 23. 16S rRNA gene sequencing process <ul><ul><ul><ul><ul><li>Bacterial colonies </li></ul></ul></ul></ul></ul>DNA extraction PCR product cleanup PCR Cycle sequencing Sequencing product cleanup Sequence detection Data assembly and analysis
    24. 24. PCR of 16S rRNA gene sequence <ul><ul><li>Universal primers were selected to amplify 1,500bp 16S rRNA gene sequence. </li></ul></ul><ul><li>Black : conserved regions </li></ul><ul><li>White : variable regions </li></ul>
    25. 25. PCR optimization Figure1A .Non-specific primer- dimmer formation seen. No desired 1,540 bp band present. 1,000 500 100 1,000 500 100 Cycling Conditions: Cycling Conditions: <ul><li>Data showing results when PCR conditions were not optimized: </li></ul>Figure 1B. Desired 1,540 bp band is present, but still non-specific bands seen. 1,500 1,500 Cycles Temp. Time 1 95 ºC 5min 35 94 ºC 30sec 52 ºC 20sec 72 ºC 2min 1 72 ºC 10min Cycles Temp. Time 1 95 ºC 5min 35 94 ºC 40sec 56 ºC 20sec 72 ºC 2min 1 72 ºC 10min
    26. 26. PCR optimization Cycles Temp. Time 1 95 ºC 5min 35 94 ºC 40sec 60 ºC 20sec 72 ºC 2min 1 72 ºC 10min
    27. 27. 16S rRNA gene sequencing process <ul><ul><ul><ul><ul><li>Bacterial colonies </li></ul></ul></ul></ul></ul>DNA extraction PCR product cleanup PCR Cycle sequencing Sequencing product cleanup Sequence detection Data assembly and analysis
    28. 28. Purification of PCR product <ul><ul><li>Amplification product was diluted 1:6 (or more depending on band size) with PCR grade water. </li></ul></ul>
    29. 29. Optimization of PCR product purification step <ul><li>Sequencing with undiluted PCR product generated very poor data with high background noise and sequences not being able to asseble into one contiq. </li></ul>
    30. 30. Optimization of PCR product purification step <ul><li>Dilution of PCR product to 1:2 generated good quality but shorter sequences (~300bp). Dilution of PCR product to1:4, 1:6 and 1:8 resulted in very good quality long (>500bp) sequences . </li></ul>
    31. 31. 16S rRNA gene sequencing process <ul><ul><ul><ul><ul><li>Bacterial colonies </li></ul></ul></ul></ul></ul>DNA extraction PCR product cleanup PCR Cycle sequencing Sequencing product cleanup Sequence detection Data assembly and analysis
    32. 32. Sequencing of PCR product <ul><ul><li>To sequence 16S rRNA gene several universal primers were selected </li></ul></ul>Black : conserved regions White : variable regions
    33. 33. Sequencing of PCR product (cont.) <ul><ul><li>Cycle-Sequencing performed using Di-Deoxy terminators (Sanger method): </li></ul></ul>
    34. 34. Sequencing of PCR product (cont.) <ul><ul><li>When a ddNTP is incorporated, further chain elongation is blocked and this results in a population of truncated products of varying lengths. </li></ul></ul>
    35. 35. 16S rRNA gene sequencing process <ul><ul><ul><ul><ul><li>Bacterial colonies </li></ul></ul></ul></ul></ul>DNA extraction PCR product cleanup PCR Cycle sequencing Sequencing product cleanup Sequence detection Data assembly and analysis
    36. 36. Purification and detection of sequencing product <ul><ul><li>Sequenced product was purified with Big Dye Xterminator purification kit. </li></ul></ul><ul><ul><li>By capillary electrophoresis (ABI PRISM Genetic analyzer) </li></ul></ul>
    37. 37. 16S rRNA gene sequencing process <ul><ul><ul><ul><ul><li>Bacterial colonies </li></ul></ul></ul></ul></ul>DNA extraction PCR product cleanup PCR Cycle sequencing Sequencing product cleanup Sequence detection Data assembly and analysis
    38. 38. Sequence assembly and analysis (cont.) <ul><ul><li>Analysis and editing of sequences was done using Lasergene DNASTAR software </li></ul></ul>
    39. 39. Comparisment of assembled 16S rRNA sequences with database library <ul><ul><li>Genbank http://www.ncbi.nlm.nih.gov/ </li></ul></ul><ul><ul><li>RIDOM h ttp://rdna.ridom.d e/ </li></ul></ul><ul><ul><li>EMBL http://www.ebi.ac.uk/embl/ </li></ul></ul><ul><ul><li>leBIBI </li></ul></ul><ul><ul><li>http://umr5558sudstr1.unilyon1.fr/lebibi/lebibi.cgi </li></ul></ul>
    40. 40. Comparisment of assembled 16S rRNA sequences with database library <ul><li>Genbank database </li></ul>
    41. 41. Comparisment of assembled 16S rRNA sequences with database library (cont.) <ul><li>LeBiBi program combines similarity search tools in the sequence databases and phylogeny display programs </li></ul>
    42. 42. Identification criteria ≥ 99% sequence similarity ≥ 97% and < 99% of sequence similarity ≥ 95% and <97% of sequence similarity Species level Genus level Family level
    43. 43. Identification criteria
    44. 44. Validation of 16S rRNA sequencing <ul><ul><li>129 isolates consisting of ATCC strains and known bacterial strains were tested with 16S rRNA sequencing </li></ul></ul>No. of known strains tested Identified to genus level Identified to species level 129 129(100%) 127 (98.4%)
    45. 45. 16S rRNA Sequencing For Gram-positive Rod Identification
    46. 47. 16S rRNA sequencing for Gram-positive rod identification <ul><li>Identification results of both methods were compared and summarized </li></ul>Method of identification Nr. of organisms tested ID to species level ID to genus level only ID to Family level only Incorrect ID No ID 16S rRNA sequencing 47 45(95.8%) 1(2.1%) 1(2.1%) 0 0 Phenotypic identification 47 15 (31.9%) 14(29.8%) 0 8(17.0%) 10 (21.3%) Focus Lab. identification 33 19(57.6%) 8 (24.2%) 0 5(15.2) 1(3.0%)
    47. 48. Investigation of isolate with discrepant results <ul><li>In-house phenotypic methods: Aerobic Gram-positive rods. </li></ul><ul><li>16S rRNA gene sequencing : Corynebacterium tuberculostearicum </li></ul><ul><li>Reference laboratory methods : Propionibacterium species </li></ul><ul><li>Common characteristics: pleomorphic Gram –positive rods, form small colonies, aerobic to facultatively anaerobic, oxidase negative, catalase positive, nitrate reductase variable. C. tuberculostearicum produces tuberculostearic acid, but it is not included in routine biochemical panels </li></ul>
    48. 49. Investigation of isolate with discrepant results <ul><li>Phylogenetic analysis results showed that isolate clustered with C. tuberculostearicum reference sequences ( ≥99% of sequence similarity ) and had only distant relationship with Propionibacterium species (84% - 86% of sequence similarity) </li></ul>
    49. 50. Investigation of isolate with discrepant results
    50. 51. Lactobacillus sp . versus Clostridium tertium <ul><li>Phenotypic identification: </li></ul><ul><li>MCR: Lactobacillus species </li></ul><ul><li>16S rRNA gene sequencing and Reference laboratory ID: Clostridium tertium </li></ul>
    51. 52. Lactobacillus sp . versus Clostridium tertium <ul><li>Common characteristics: slender gram-positive rods, Alpha hemolytic, grow in aerobic conditions, catalase neg., oxidase neg. </li></ul>Clostridium tertium Lactobacillus sp.
    52. 53. Corynebacterium sp versus Actinomyces neuii <ul><li>Phenotypic identification: </li></ul><ul><li>Diphtheroids - [Corynebacterium species] </li></ul><ul><li>16S rRNA gene sequencing and Reference laboratory ID: Actinomyces neuii </li></ul><ul><li>Isolate characteristics: small gram-positive rods, non-hemolytic, grow in aerobic conditions, catalase pos., oxidase neg., CAMP test variable, urea neg. CoryneAPI : 1 case – not performed, 2 cases – no identification) </li></ul>
    53. 54. Corynebacterium sp versus Actinomyces neuii <ul><li>Phylogenetic analysis results revealed that Actinomyces sp share only 82%-86% of sequence similarity to Corynebacterium sp </li></ul>
    54. 55. Corynebacterium sp versus Actinomyces neuii
    55. 56. Taxonomic reclassification of bacteria <ul><li>Phenotypic methods and reference lab. methods : Bifidobacterium Sp. </li></ul><ul><li>DNA sequencing : Alloscadovia omnicolens ( separated from Bifidobacterium sp as new species in 2007) </li></ul><ul><li>Exhibits the same phenotypic and biochemical characteristics as Bifidobacterium sp, but taxonomic positioning based on DNA sequencing shows that isolate belongs to a novel species and genus. </li></ul>
    56. 57. Labor intensiveness and Turn-around time assessment of sequencing Steps Procedure Labor Time (hands-on) Waiting time (machine time) 1 Extraction of bacterial DNA 5 min 10 min 2 Master mix preparation, PCR amplification 10 min 3 hours, 30 min 3 Analysis of the PCR product. Loading, running, and examining gel. 10 min 20 min 4 Dilution of PCR product and Sequencing step 15 min 2 hours, 30 min 5 Purification of PCR products 5 min 32 min 6 Assembling capillary tray for sequencing, loading tray to the Genetic analyzer. 5 min 1 hour 7 Sequence assembly, editing, database search 10 min   9 Reporting of results. 5 min     Total labor time 1 hours 5 min/ per 1 isolate (add 10 min to each additional isolate) 8 hours and 35 min
    57. 58. Cost evaluation of 16S rRNA sequencing *- cost is subjected to variations depending on organism. Cost of consumables Cost of labor Total cost per test 16S rRNA sequencing kits, materials, reagents (instrumentation not included) : $30.34 $35.0 $65.34 Phenotypic identification * materials, reagents, biochemicals, medium, commercial tests (API, Rapid ANA) (instrumentation not included) : $15.0 $15.0 $30 $110 - if isolate is sent out to reference lab. for identification
    58. 59. Evaluation of first 500bp sequence for bacterial identification <ul><ul><li>Of 30 Gram-positive rods tested, the first 500bp gave 100% (30 of 30) match to the same genus and 83% (25 of 30) match to the same species as full sequence </li></ul></ul>Region Genus level Species level Sequencing primers ≥ 500bp 30 (100 %) 25 (83.3%) 8F, 357R, 531R 1,500bp 30 (100%) 30 (100%) 8F, 515F, 357R, 531R,1104F, 787R
    59. 60. Conclusion <ul><li>16S rRNA sequencing is more accurate, faster and, yet, cost effective comparing to conventional identification methods, especially when used for identification of Gram-positive rods with ambiguous biochemical profiles or slow growers. </li></ul><ul><li>Furthermore, this technique is applicable to other organisms such as Mycobacteria , of which identification is not routinely performed in most of clinical microbiology laboratories due to requirements of special expertise and equipment. </li></ul>
    60. 61. Conclusion <ul><li>In addition to this, the workflow of 16S rRNA sequencing can be defined and standardized for all bacterial isolates, independently of their individual phenotypic characteristics and growth requirements within the set timeframe </li></ul><ul><li>Interpretation of nucleotide sequence data is more objective and straightforward then interpretation of biochemical results, most of the sequence editing programs are preset to search for ambiguous nucleotides making editing job fast and easy. </li></ul><ul><li>Raw data generated from 16S rRNA sequencing (sequence electropherograms) can be stored in electronic format and easily retrieved for future laboratory or epidemiological studies. </li></ul>

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