ANNE_S MIDTERMS [CLBACT LEC] MT_25 - Y3T1.pdf

CLBACT LECTURE NOTES / MIDTERM WEEK 7: CLINICAL MICROBIOLOGY WORKFLOW AND GENERAL CONCEPTS IN BACTERIAL IDENTIFICATION MICROBIOLOGY LABORATORY TESTING PROCESS IDENTIFICATION METHODS The identification of a bacterial isolate analysis of information gathered from laboratory tests that provide charac...

CLBACT LECTURE NOTES / MIDTERM WEEK 7: CLINICAL MICROBIOLOGY WORKFLOW AND GENERAL CONCEPTS IN BACTERIAL IDENTIFICATION MICROBIOLOGY LABORATORY TESTING PROCESS IDENTIFICATION METHODS The identification of a bacterial isolate analysis of information gathered from laboratory tests that provide characteristics profiles of bacteria. The tests and the order in which they are used for organism identification are often referred to as an identification scheme or workup of the organism ● Tests done on each identification method are called identification scheme or work up ● Determining the clinical significance of a particular pathogen ○ Kung yung microorganism ba na present is totoong pathogen or baka contaminant lang or normal flora ng patient ● Guiding physician care of the patient through presumptive and final identification methods ○ It gives an overview kay doctor kung ano yung present dun sa specimen, depende ano specimen ipapasa depende if physical exam sinasabi ni doc kung ano symptoms ○ Upon receiving, we give presumptive identification for them to an immediate therapeutic drug to the patient ● Determining whether laboratory testing for detection of antimicrobial resistance is warranted ○ Aid in diagnosis in treatment ● Determining the type of antimicrobial therapy that is appropriate ● Determining whether the antimicrobial susceptibility profiles are unusual or aberrant for a particular bacterial species ● Determining whether the infecting organism is a risk for other patients in the hospital, the public, or laboratory workers ○ As a form of infection control pag may tumubo dyan na microorganism fungi or virus malalaman as a healthcare worker if we are at risk in getting that and also in public, dikit yung public health on how we treat diseases ● Collecting epidemiologic data to monitor the control and transmission of organisms MICROBIOLOGY LABORATORY TESTING PROCESS (CONVENTIONAL) WORKFLOW Table 1. Conventional Workflow Time Test 20 30 ● Generation time for most clinically minutes relevant bacteria ● Growth based test usually require Generation hours of incubation before the Time presence of an end product can be measured 18 24 ● Required time for most conventional hours identification schemes ● Time before the test can be Growth accurately interpreted Based ● Time it take for a bacteria to reproduce into 2 bacteria ● ● Note : Generation time / Doubling time ○ Time it takes for a bacterium to reproduce into 2 -0 bacteria Growth based test ○ Dependent on the generation time Gram Pot Bacili cocci ● ● ● ● ● ● ● ● gram negative Bacilli cocci Presumptive report: four possible groups ~ gram pos [(1) bacilli, (2) cocci], gram neg [(3) bacilli, (4) cocci] ○ Crucial ‘to para mabigyan ng drugs yung patient released after reading the gram stain (presumptive report) Specimen processing after report do primary media and inoculation ○ PRIMARY MEDIA: blood agar plate (broth media or plate) Next, i-examine yung primary culture features (how it look like check for colonies then do additional testing depending on the features you have seen and can do subcultures then antibiotic susceptibility then release final identification then release final report tapos notify physician then sya yung mag interpret ng need gawin sa patient Primary media: BAB (blood agar plate) - commonly used First specimen collection: always take note of the basic principles in handling crucial sya sa microbiology.BkYou can contaminate the specimendapat sterile mga gamit kasi baka wala naman pathogen tas na contaminate mo tapos bibigyan ni doctor ng drugs si patient kahit wala palang virus/bacteria sa katawan Some laboratories have bar code and rfid automated na pag nagrereceive ka ng specimen para kang nag cacashier, punch ng bar code lang. Nasa information system na sya ng laboratories pero sa Philippines wala masyado kaya marami clerical errors NMS [NO MICROORGANISM SAME (?)] BASIC PRINCIPLES OF SPECIMEN COLLECTION AND HANDLING Specimens should be collected from the appropriate site for recovery of suspected pathogens. BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 1/32 ● Specimens should be collected prior to the administration are minimized. Additionally, antigen-antibody reactions can antibiotics be employe ● Collection techniques must be clearly identified and should ● Not based on the bacterial growth avoid contamination with normal flora ● Based on the enzymatic activity and antigen ● The quantity of specimen collected should be adequate for antibody reaction of bacteria all requested cultures and testing ● Kahit hindi pa ganoon karami ang bacteria na ● Specimens should be transported and processed in a represent, we can identify na through enzymes and timely manner antigen - antibody interactions ● Transport media should be utilized to ensure viability of organisms when processing may be delayed ● Specimens should be accurately labeled to include patient identification, date and time of collection, and the source ● NOT GROWTH BASED TEST of the specimen ● The laboratory should be notified in advance if an unusual pathogen is suspected to ensure appropriate collection and handling ● The generation time for most clinically relevant bacteria is 20 to 30 minutes, PHENOTYPIC CRITERIA / APPROACH ● Growth-based tests usually require hours of incubation ● Are based on observable physical or metabolic before the presence of an end product can be measured. characteristics of bacteria through analysis of gene Many conventional identification schemes require 18 to 24 products rather than through the genes themselves. hours of incubation, or longer, before the tests can be ● We do not test the genes but the gene products such as accurately interpreted proteins in the presence of our enzymes and aids in the ○ Generation time = doubling time : It is the time it takes metabolism of the bacteria for bacteria to reproduce two (2) daughter cells from ● Classic approach to bacterial classification and one mother cell so basically yung mother cells identification magiging two (2) daughter cells. Conventional time MOST COMMONLY USED PHENOTYPIC CRITERIA naka base dun dahil need pa sila i-incubate before ● Microscopic morphology and staining characteristics you proceed to the different tests to be done. ○ Gram stain, acid fast bacilli MICROBIOLOGY LABORATORY TESTING PROCESS ● Macroscopic (colony) morphology, including odor and ← (RAPID) WORKFLOW be pigmentation ● Rapid method is one that provides results the② same day can in ○ Growth in the medium seen that the test was inoculated. eyes ○ Odor ● Alternatively, the definition may be more precise, whereby ○ Color rapid is only used to describe tests that provide results ○ Size within 4 hours of inoculation. ○ Shape ● It is important to note that rapid identification still requires ○ Margin overnight incubation of culture media from the primary ○ “Smelling is strongly discouraged in clinical specimen. setting” ○ Alternative sya sa conventional workflow na need Rapid ● Environmental requirements → : Inoculation18-24 hours. Dito, it can provide results within 4 hours workflow ○ O2 primary ○ CO2 within inoculation lagi need primary culture dahil dun media culture ○ Temperature mo kukunin specimen for further testing, it can be a ■ Certain bacteria would grow at 37°C (body variation of conventional testing, you can decrease temperature) the test substrate(?) or increase concentration of ■ Some have the ability to grow at higher and media lower temperature ○ Next is unconventional: di naka base sa bacterial ■ Campylobacter jejuni - grow at 42°C growth pero tatandaan yung mga non-growth base ■ Yersinia enterocolitica - grow at 0°C test second general approach naka base sa products ○ Thioglycollate broth: enzymatic or metabolic activity mga antigen ■ Aerobic antibodies depending sa antigen present sa bacteria ■ Anaerobic substrate medium concentration TWO GENERAL APPROACHES: f ■ Microaerophilic 1. Vary the conventional testing approach by decreasing ● Resistance or susceptibility to antimicrobial agents \ the test substrate medium volume and increasing the surface ○ Susceptibility testing concentration of bacteria in the inoculum have ○ layer Scenario: You perform gram staining but you're in 2. Unique or unconventional substrates by choosing if you use doubt towards the result you obtain. particular substrates based on their ability to detect doubts ○ You can use antimicrobial agents to verify if the result enzymatic activity at all times. This is not a growth-based ! test! you obtain is correct test so delays caused by depending on bacterial growth ● Obligate aerobe - needs oxygen Based on Enzyme and antigen antibody Reactions Facultative anaerobe ^ 1 antimicrobial , - BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 2/32 ● ● ● ● Aerotolerant anaerobe Microaerophilic - little oxygen requirement ■ Thioglycollate broth: ● Req CO2 Neisseria gonorrhoeae spp. Haemophilus spp. You can use antimicrobial agents to verify if the result you obtain is correct. GRAM POSITIVE GRAM NEGATIVE Use vancomycin for gram-positive organisms. Because gram positive organisms are susceptible except Moraxella, Virsa (vancomycin resistant Staphylococcus aureus). Even though they are gram-positive, they resistant to vancomycin are Generally, all gram-positive bacteria are susceptible to vancomycin Use colistin and polymyxin to verify if it is gram-negative (susceptible). Setting” can create aerosols ○ Environmental requirements for growth ■ O2 ■ CO2 ■ Temperature ○ Resistance or susceptibility to antimicrobial agents ○ Nutritional requirements and metabolic capabilities ○ Biochemical reactions including enzymatic reactions or chemical profiles PHENOTYPIC IDENTIFICATION METHODS ● Types of Enzyme-Based Tests: ○ Catalase - test for presence of catalase sa bacteria ○ Oxidase - cytochrome oxidase ○ Indole - tryptophanase ○ Urease - urea ○ PYR - test for l-pyroglutamyl-aminopeptidase ○ Hippurate- hippuricase bacteria TYPES OF ENZYME BASED TEST — Catalase Test for catalase Oxidase Cytochrome oxidase Indole Tryptophanase Urease Urease / Urea hydrolysis test PYR Hippurate ● urine normal secretion L-pyroglutamyl-aminopeptidase (PYR-enzyme) — MICROBIOLOGY LABORATORY TESTING PROCESS (PHENOTYPIC) IDENTIFICATION ● Phenotypic criteria are based on observable physical or metabolic characteristics of bacteria through analysis of gene products rather than through the genes themselves. The phenotypic approach is the classic approach to bacterial classification and identification. ○ Naka base sa physical and metabolic characteristics, gene products ang tinetest sa bacteria considered a classical approach ○ Stain afb - microscopic morphology ○ Aided by the naked eye yung naoobserve mo sa colony ng media mo either broth or plated test for odor pigmentation etc - macroscopic (odor not advised baka may aerosols baka mainfect ka pag inamoy mo directly done only if fully closed or dapat nasa fuse hood ka ○ Nutritional requirements : capability to metabolize a certain substance ○ Biochemical : enzymatic reaction and chemical profile ● The most commonly used phenotypic criteria include the following: ○ Microscopic morphology and staining characteristics ■ Gram stain, acid fast bacilli ○ Macroscopic (colony) morphology, including odor and pigmentation ■ Growth in the medium ■ Odor ■ Color ■ Size ■ Shape ■ Margin “Smelling is strongly discourage in clinical → Hippuricase Test for Metabolic ○ Oxidation and Fermentation ○ Amino Acid Degradation: either decarboxylation or deamination ○ Single Substrate Utilization ○ Inhibitor Profiles TESTS FOR METABOLIC PATHWAYS Oxidation and fermentation (CHO-Carboh ydrates) Reason of two tubes Test the capability of the bacteria to oxidize or ferment sugar or carbohydrate Oxidative Fermentative (OF) medium - has glucose and low concentration of peptone. Main detection of glucose 1. OF Test used 2 tubes 2. Has peptone and glucose in the medium 3. Specimen is transferred to inoculum 4. Both tubes are incubated 5. But we will put mineral oil in 1 tube to drive off oxygen. Set as barrier for medium to not let any oxygen enter the tube 6. We check if one is anaerobic and one is aerobic. That’s why we have 2 tubes 7. The 1st tube is for aerobic, and the 2nd is for anaerobic with mineral oil. To prevent oxygen from entering the medium 8. Medium has 2 indicators: Andrade’s indicator and Bromocresol Purple (BCP) 9. Bromocresol purple is usually used (color change of green to yellow) 10. If the bacteria utilized the glucose the environment will be acidic turning from ↳ Brome cresol green to yellow & BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 3/32 11. 3 possible results: a. Oxidative/ Nonfermenter - First result turned into yellow = it was able to utilize glucose in the presence of oxygen (aerobic). While tube 2nd , anaerobic has no color change, therefore it wasn’t able to use glucose in the absence of oxygen i. Can use glucose in the presence of oxygen ii. Aerobic can only ferment/ utilize b. Fermentative/Fermenter - in the presence or absence of oxygen it can utilize glucose i. If it can utilize glucose in both absence and presence of oxygen c. Non - utilizer - it didn’t utilize glucose even in the presence or absence of oxygen. It cannot utilize glucose at all Inhibitor profiles Notable na antibiotics Inhibits bacteria Streptococci: gram-positive), Vibrio Optochin and Bile solubility antimicrobial agent Useful in Streptococcus pneumoniae (not in a b c d groups) Bile esculin hydrolysis - useful for Enterococci N O B E Ethanol survival test - even in the presence of ethanol, it can resist/ grow Use for Bacillus MICROBIOLOGY LABORATORY TESTING PROCESS INITIAL SPECIMEN WORKUP Amino acid degradation 1. Decarboxylation - cleaves carboxyl mino acid group from Amino Acid (AA) (convert amino acids to amines). Happens to Anaerobic to Amines indicator process Red Phenol a. Moeller’s Test - has 3 amino acids: Bromo cresol mnemonics 1- LOA : Lysine, Ornithine, Arginine ( Purple Figure 2. Initial Specimen workup may dalawang indicator na Yellow / Purple ) ginagamit: phenol red or SELECT AND INOCULATION OF TEST bromocresol purple (BCP) YELLOW Decarboxylation a. If we isolate bacteria, we choose certain test included TO PURPLE (able to decarboxylate Purple for the workup depending on the presumptive magiging purple kapag phenol red, identification Final Product magiging red ang final color product b. If its gram positive, we will include certain test for the Red kapag na-cleave ni bacteria yung workup, if it is gram negative, it differs from gram carboxyl group) positive 2. Deamination - cleaves amine group amines c. Incubate first in the primary medium. After streaking → from amino acid from AA to Amino then incubation, you can now identify Acid a. PDA - phenylalanine deaminase d. For us to be able to produce pure culture, inoculate Agar (contains 10% ferric chloride resulting first to possible color of green) e. BATTERY OF TEST - test included in the workup or Phenylalanine deaminase test the identification scheme (choose those compatible gumagamit ng ng PDA AGAR LIA for our presumptive report) Ask 9 (lysine iron agar) - merong lysine ! tapos incorporated with dextrose INCUBATION FOR SUBSTRATE UTILIZATION 0.1% kapag able si microorganism a. Metabolic pathways, enzymes to deaminase lysine (positive color will be purple to yellow) b. Depend on the bacterial multiplication c. Growth based test vs Non growth based test Lysine IA (LIA) (contains 0.1% dextrose) - - to = = :{ Friend I Single substrate utilization “Until here lang ang metabolic pathways” A- antibiotic - If kaya ba ni bacteria mag grow sa presence ng citrate, alanate, etc acetate Inhibitor profile: maraming examples notable and antibiotics if kaya ba ng antibiotics mag grow ng certain bacteria Citrate - if the bacteria can utilize citrate Malonate - if the bacteria can utilize malonate oangwwat Acetate - if the bacteria can utilize acetate as Bacteria specific a carbon source Addtl. NaCl - Enterococci (group d DETECT OF METABOLIC ACTIVITY (SUBSTRATE UTILIZATION) a. b. Colorimetry - color change in the mediu Fluorescence - pH change Substrate fluorophore complex c. Turbidity test d. Optical density - measure by spectrophotometer or nephelometry e. Detect various photometers BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 4/32 ● ANALYSIS OF METABOLIC PROFILES MICROBIOLOGY LABORATORY TESTING PROCESS (GENOTYPIC) IDENTIFICATION Involve characterization of some portion of a bacterium’s genome using molecular methods for DNA or RNA analysis. (nucleotides) Genome of the microorganism - definitive approach dna and rna our nucleotide which is present in the genome the microorganism, test for specific gene molecular bio technique ginagamit para sa nucleotide This usually involves detecting the presence of a gene, or a part thereof, or an RNA product that is specific for a particular organism. In principle, the presence of a specific gene or a particular nucleic acid sequence unique to the organism is interpreted as a definitive identification of the organism. ● ● ● ● MOLECULAR DIAGNOSTIC TECHNIQUES ● based on the consistent and somewhat predictable nature of DNA and RNA. The nucleic acid–based methods are classified into the following categories: 1. Hybridization 2. Amplification 3. Sequencing and enzymatic digestion 4. MICROBIOLOGY LABORATORY TESTING PROCESS MOLECULAR DIAGNOSTIC TECHNIQUES ● ● ● STEPS ON PROBE 1. 2. 3. 4. ● NUCLEIC ACID HYBRIDIZATION Based on the ability of two nucleic acid strands with complementary base sequences to bond (H. bond) specifically with each other and form a double- stranded molecule, called a duplex or hybrid. To identify the presence of an organism, hybridization assays involve duplex formation between two nucleic acid strands: ● ● 2. Target (Template) - sequence that will be identified (immobilized and suspended in the solution Probe - single-stranded RNA or DNA oligonucleotide labeled with a reporter chemical (may label fr) (radionuclide or fluorescent particle) amines AA ● Attached with certain label → ● If we have DNA or RNA strand then denatured, the hydrogen bonds will break between them; ○ Then a complementary strand base sequence will be attached. Therefore, called as Hybridization process ● It test for the ability of two nucleic acids to stand with their complementary sequences to create a bond from og strand and its complementary bases (hydrogen bond) if nag fform ng double stranded duplex yun or hybrid dna strand/ rna strand na nakadikit sa isang complementary base ● Single strand DNA and RNA - will form duplex or ↳ double hybrid Production and labeling of single-stranded nucleic acid probe a. Label probe with reporter chemicals Preparation of single-stranded target nucleic acid a. Genome sequence to be identified in the microorganism b. Reporter chemicals to be used: i. Radionucleotide ii. Fluorescent particles Mixture and hybridization of target and probe nucleic acid Detection of hybridization Examples: (SNOWDROP) 1. Southern Blot (DNA) ● Solid support: agarose gel electrophoresis ● Uses gel electrophoresis, solid support hybridization 2. Northern Blot ● Uses gel electrophoresis, solid support hybridization ● Solid support: agarose gel electrophoresis 3. 4. TWO COMPONENTS OF DUPLEX/HYBRID 1. They will form hydrogen bonds with its complementary base sequence Two complementary bond for Adenine - THYMINE Three hydrogen bonds for Guanine - Cytosine Hybridization depends on the hydrogen bonds formed if it will form duplex or hybrid In-situ hybridization (liquid medium) ● In situ = in place or in position → direct labeled ● Directly in tissue with labeled probes then detect the tissue attached ● Done in paraffin embedded tissue PNA FISH (under in-situ) Peptide Nucleic Acid Fluorescence In Situ Hybridization ● Instead of DNA or RNA, the probe used is PNA probe to longershelf > due r ○ Advantage: it cannot be degraded by nucleases and proteases unlike DNA and RNA that can be degraded ○ Longer shelf life = cannot be degraded - strand Ttarget microorganism ● in short amount of time NUCLEIC ACID AMPLIFICATION TEST (NAAT) Based on increasing the target microorganism’s nucleic acid in a sample in a short amount of time. (this one can be detected by various methods PCR and non PCR BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 5/32 life ● ● ● amplification) This increase can be detected by various methods: Slow grower orgs Non cultivable orgs Sterile body fluids ○ Dahil sobrang konti lang ng microorganisms such as pleural cavity, csf, peritoneal cavity so sa mga membranes mahirap makapasok yung microorganisms so NAAT helps increase the concentration of the microorganisms. ○ Inaamplify nya yung mga target nucleic acid ng microorganism in a short amount of time POLYMERASE CHAIN REACTION (PCR) ● ● ● ● Remember A simple in vitro chemical reaction that permits the synthesis of essentially limitless quantities of target nucleic acid sequence Ba isang PCR cycle nangyayari itong tatlong steps na ito So pwedeng 30 to 40 cycles Steps: Steps 1.! Denaturation - separates double-stranded DNA, happens at 90-96 degree celsius at 20-60 seconds a. Most essential is the DNA 2. Annealing - anneals primers to target DNA a. 50 to 70 degree celsius at 20-90 seconds 3. Extension - synthesizes new strands of DNA a. 68 to 75 degree celsius at 10-60 seconds netunperatme + ● ● ● Arbitrary primed PCR Quantitative PCR Digital PCR Probe is for hybridization while prime is for amplification NON - PCR AMPLIFICATION TEST ● Nucleic Acid Sequence-Based Amplification (NASBA) ○ 3 enzymes: Focus ■ AMV RT ■ Ribonuclease H (RNase H0) ■ T7 RNA polymerase ● Transcription Mediated Amplification (TMA) ○ 3 enzymes: ■ RT (Reverse transcriptase ■ RNase H ■ T7 RNA polymerase NASBA and TMA are both isothermal, single temp at 41 degree celsius ● Exploit signal detection rather than amplification of the target nucleic acid ● Amplifying ONLY the signal not the target SIGNAL AMPLIFICATION METHODS ● Exploit signal detection rather than amplification of the ↳ prime !! target nucleic acid more ○ Amplifying ONLY the signal not the target base sequence ○ For labels of the signal amplification methods we have AP = Alkaline phosphatase ont-nty.me Bwzmm-nmkmm understand DIFFERENT KINDS OF SIGNAL AMPLIFICATION METHODS ● ● ● REAL - TIME PCR (KINETIC PCR/HOMOGENOUS PCR) ● ● ● ● ● Real-Time PCR (also known as kinetic PCR/Homogenous PCR) Amplicons which are detected real-time as they are accumulated after each cycle Mostly other PCR detects amplicons at the end, dito sa real time nadedetect niya yung amplicons after each cycle So agad agad si real time Different Detection Variation: ○ 5 nuclease assay (Taq man) ○ Dual probe FRET (fluorescence resonance energy transfer) ○ Molecular beacon ○ Scorpion primer ○ Intercalating dye ■ Example: SYBRE Green RT-PCR (Reverse transcriptase PCR or RNA PCR) Other examples of PCR (mentioned by prof): ● Multiplex PCR ● Nested PCR Branched DNA (bDNA) detection ○ Yung signal is yung bDNA ○ Has amplifier probes that bind to preamplifier probes (which are the branched DNA) Hybrid Capture Technology ○ Detect the hybrid ○ RNA probe bind to target DNA Cycling Probe Technology ○ Also runs isothermic ○ Detects the signal Chimeric probe which is composed of DNA - RNA - DNA PCR COMPONENTS ● ● ● ● ● ● Target (Template DNA) - serves as our target base sequence for PCR Oligonucleotide Primers - used to start synthesis of new DNA strands (thermostable dna polymerase) ○ MgCl2 = cofactor of taq polymerase Heat Taq polymerase (Thermostable DNA polymerase) aids the synthesis of new strands of DNA (thermostable dna polymerase— after kumabit ni prime sa target Magnesium Chloride (MgCl2) - required for function or cofactor of polymerase/ Taq polymerase Buffer - ensures proper conditions of pH for polymerase tris-HCL (trisaminomethane) and salt (KCL) ph at 8.3 para mag perform better si polymerase Deoxynucleotides - used by polymerase to synthesize new DNA strands BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 6/32 ● ● ● ● Thermal cycler - instrument that rapidly allows the heating/ cooling depending on the PCR cycle steps PCR Amplicons - product of Real pcr (new DNA strands) should be stored at -20 degree celsius or analyzed immediately ○ Dito na yung part kung saan imemeaseure natin or quantify if may na produce na PCR amplicons or the new DNA strands ○ Kapang negative meaning walang amplicons ○ Normal pcr take = 30-40 cycles NUCLEIC ACID SEQUENCING Involves methods that determine the exact nucleotide sequence of a single gene or gene fragment obtained from an organism ← It can also be the whole genetic make-up of a certain organism a. ● ENZYMATIC DIGESTION AND ELECTROPHORESIS OF DNA FRAGMENTS ● DIFFERENT WAYS OF DOING SEQUENCING ● ● Conventional Sequencing Pyrosequencing ○ It is a traditional and rapid test sequencing technique of a certain gene or fragment wherein it uses 20-50 base long sequences for its primer suitable only to short gene fragments or short sequences. ○ Mas ginagamit itong pyrosequencing, mas mabilis gawin ○ Which uses enzymes such as: 1. DNA polymerase 2. ATP sulfurylase 3. Luciferase 4. Apyrase 5. APS (Adenosine Phosphosulfate) 6. Luciferin NEXT - GENERATION SEQUENCING ○ Increased speed, newer and enhanced accuracy, short or long ito yung mas ginagamit *Mas ginagamit ang metagenomics sa environmental studies wherein may mga mixed population ng organisms b. To identify nonculturable and mixed populations organisms c. Also 1% lang ang nacculture sa lab so metagenomics helps to detect these microorganisms Nucleic Acid Electrophoresis ○ 2 most common buffers: ■ Tris Acetate ■ Trisborate ● ● ● ● Are done to provide valuable information for the diagnosis and control of infectious diseases. It is accomplished using any of a number of enzymes known as restriction endonucleases. Each specific endonuclease recognizes a specific nucleotide sequence (usually 4 to 8 nucleotides in length), known as the enzyme’s recognition or restriction site. Restriction sites are often palindromic sequences; in other words, the two strands have the same sequence, which run antiparallel to one another. Once the recognition site has been located, the enzyme catalyzes the digestion of the nucleic acid strand at that site, causing a break, or cut, in the nucleic acid strand. ○ Kapag na cut na yung nucleic acid strand it will then be allowed to undergo Gel electrophoresis or the nucleic acid electrophoresis Restriction Patterns can be differentiated by Restriction Fragment (Length or Locus) Polymorphism (RFLP) Yung buong process na ito is called: Restriction Enzyme Analysis (REA) ○ Ginagamit din siya sa sequencing pero dito fragments ang tinetest ○ Also used to provide information for control and detection DNA MICROARRAYS AND NANOARRAYS ○ Grouping of DNA molecules at a micron and nano like CHIP sa computer level → just ○ DNA/gene chip - evaluates gene expression of the entire organism (tawag dun sa group ng dna molecules used to evaluate gene expression of an entire organism ■ Enzymatic digestion - conduct electrophoresis Book after digesting the gene you produce dna find fragments *restrictions endonuclease* dapat definition yung mga restriction site, they run antiparallel to each other) { PROTEOMICS ○ Studies the proteins of organisms 1. MALDI-TOF MS 2. Metagenomics (e.g those in the gut) MICROBIOLOGY LABORATORY TESTING PROCESS APPLICATION OF NUCLEIC ACID-BASED METHODS ● Direct detection of microorganisms in patient specimens ● Identification of microorganisms grown in culture ● Characterization of microorganisms beyond basic identification CHARACTERIZATION OF MICROORGANISMS BEYOND IDENTIFICATION ● Situations exist in which characterizing a microbial pathogen beyond identification provides important information for patient management and public health. BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 7/32 ● ● ! ILION apter Although various phenotypic methods have been able to provide some of this information, the development of nucleic acid-based testing has greatly expanded the capability to generate this information in the diagnostic setting in a more timely fashion. This is especially true with regard to antimicrobial resistance and strain relatedness among bacteria Table 8-4: Examples of Methods to Determine Strain Relatedness METHOD Plasmid analysis ADVANTAGES / LIMITATIONS Simple to implement but cannot often discriminate because many bacterial species have few or no plasmids. Multilocus enzyme electrophoresis Provides only an estimate of overall genetic relatedness and diversity (protein-based). Multilocus sequence typing Data are electronically portable and used as a non-culture-based typing method; labor intensive and expensive. Pulsed-field gel electrophoresis Highly discriminatory but it is difficult to resolve bands of similar size and interlaboratory reproducibility is limited. Randomly amplified polymorphic deoxyribonucleic acid (DNA) Highly discriminatory power but poor laboratory reproducibility die to short random primer sequences and low polymerase chain reaction (PCR) annealing temperatures. Repetitive Manual system: Useful for strain sequence-based PCR typing, but low rates of interlaboratory reproducibility; suboptimal turnaround times (TATs) for both manual and automated systems. Automated system: Increased reproducibility and decreased TATs. Ribotyping and PCR ribotyping ● ○ ○ ○ ○ ○ Repetitive Palindromic Extragenic Elements Polymerase Chain Reaction Arbitrary PCR ■ Approximately 10 nucleotides long (Rep-PCR) Multilocus Variable Number of Tandem Repeat Analysis ■ Tests repetitive DNA sequences Multilocus Sequence Typing Table 8-1: Examples of Automation and Instrumentation Available for the Molecular Microbiology Research and Clinical Laboratory INSTRUMEN MANUFACTU T RER Traditional Thermal Cyclers Real-Time instrument Veriti Thermal Cycler End-point thermal cycler; FDA-approve d for IVD use GeneAmp Applied PCR System Biosystems; 9700 Thermo Fisher Scientific, Waltham, MA Interchangeab le sample block modules for flexibility 7500 Fast System Applied Biosystems; Thermo Fisher Scientific, Waltham, MA IVD applications available in certain countries QuantStudio Systems Applied Biosystems: Thermo Fisher Scientific, Waltham, MA TaqMan array and OpenArray; IVD infectious disease testing Difficult to distinguish among different subtypes. STRAIN TYPING Non-amplified Typing Methods: 1. Plasmid Profile Analysis - it uses extrachormosomal molecules present in the plasmid (plasmid DNA) 2. Southern Blot - DNA particles via hybridization Restriction Fragment Length Polymorphism (RFLP) 3. Restriction Fragment Locus Polymorphism a. Enzyme used is Restriction endonuclease to identify 4-8 bases long 4. Pulsed Field-Gel Electrophoresis a. ( most used in microbiology) In gel electrophoresis 5. Multilocus Enzyme Electrophoresis (MLEE) a. Involves varying locations of DNA are being measured ○ Includes extraction of protein isolates of interest ● Amplified Typing Methods: ○ (RAPD) Random Amplified Polymorphic DNA Technique Applied Biosystems; Thermo Fisher Scientific, Waltham, MA COMMENTS CFX Systems Bio-Rad, Hercules, CA Various well formats for flexibility LightCycler Series Roche Diagnostics, Indianapolis, IN Real-time PCR platform; low-high throughput range of instruments available; infectious disease testing available SmartCycler System Cepheid, Real-time Sunnyvale, CA platform; expandable to up to 96 independent tests; BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 8/32 multiplex capacity Isothermal Instrument Sequencing Illumipro-10 Meridian Bioscience, Inc., Cincinnati, OH Automated isothermal amplification and detection; reduced hands-on time; approximately 2 minutes FDA approved 3500 Series Genetic Analyzers Applied Biosystems; Thermo Fisher Scientific, Waltham, MA CE-IVD labeled 5500W Series Genetic Analysis Systems Applied Biosystems; Thermo Fisher Scientific, Waltham, MA Next-generati on sequencing for research use only; flow chip design Semi-Autom COBAS ated Amplicor Analyzer Fully Automated COBAS AmpliPrep/ COBAS TaqMan Analyzer Roche Diagnostics; Indianapolis, IN Automated extraction and real-time PCR platform FDA-approve d testing available Widely used for viral load testing Panther System Hologic, Bedford, MA Fully automated platform with primary tube sampling to detection Endpoint and real-time transcriptionm ediated amplification FDA-approve d testing available Verigene System Nanosphere, Northbrook, IL Fully integrated microfluidic test cartridge in closed system FDA-cleared blood culture, gastrointestin al, and respiratory assays available GeneXpert and GeneXpert Infinity Cepheid, Fully Sunnyvale, CA automated, extraction, real-time detection in closed system; fully automated expanded walkaway infinity system Roche Real-time Diagnostics; PCR platform Indianapolis, IN FDA-approve d infectious disease testing available INFINITI Plus AutoGenomics, Postamplificati Analyzer Vista, CA on, microarray closed analytic system FilmArray d testing available BioFire Diagnostics Inc., Salt Lake City, UT COBAS 4800 Roche System Diagnostics; Indianapolis, IN Respiratory, gastrointestin al, and blood culture identification panel for 201 different infectious agents FDA-cleared Uses multiplex nested PCR, coupled with film-array detection Automated extraction and real-time PCR platform FDA-approve Amplificatio Plex-ID n and Mass Pathogen Spectrometr Detector y Iridica/Abbott Molecular, Abbott Laboratories, Abbott Park, IL Stand-alone or integrated system that includes extraction and processing; uses PCR platform and high-resolutio n DNA mass spectrometry Emphasize in the table: FilmArray and GenXpert check part 12 and See book Bailey’s and Scott 14th Edition BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 9/32 ● AUTOMATED APPROACH FOR DEFINITIVE IDENTIFICATION BACT/ALERT uses colorimetric detection, other fluids can be applicable here BIOCHEMICAL METHODS BIOCHEMICAL METHODS ○ Is a kind of phenotype since it only studies the gene product such as proteins, metabolic tests etc BD BACTEC Automated Blood Culture System ANALYTICAL PROFILE INDEX (API) 20E ○ ○ ○ Is a biochemical panel for identification and differentiation of members of the family Enterobacteriaceae. Can also be used for gram positive acea and gram positive cocci but mainly for the family Enterobacteriaceae Has 20 miniature wells with different chemical substrates STEPS 1. Bacterial suspension is used to rehydrate each of the wells and the strips are incubated. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. 2. All positive and negative test results are compiled to obtain a profile number, which is then compared with profile numbers in a commercial codebook to determine the identification of the bacterial species. a. Lahat ng database sa automated, duon nakalagay lahat ng profiles ng mga clinically important or known microorganisms MOLECULAR METHODS PCR I Bio fire multiplex nested pcr (panels yung nasa lower right) Has panels Multiplex nested Real time pcr Puwede rin for other fluid testing Detection of Mycobacterium tuberculosis Detect Rifampicin resistance Detect CNTG (chlamydia, neisseria, trachomatis, and gardnerella vaginalis) Film Array GeneXpert watch video ● ● ● ● ● ● ● ● Biomerieux BacT / ALERT Microbial Detection System CULTURE BOTTLES Culture bottles are not solely used for blood specimens since it can be used for other bodily fluids Anticoagulant uses SPS (Sodium polyanethol Sulfonate) Green Aerobic S Orange Anaerobic Yellow Sterile Body Fluids Has 21 microwells - 21 tests NOTE: Yung profile numbers is yung binary code and then from there dun ma-determine yung commercial code or the octal code Binary code contains 21 digits (As seen in the table the binary code is: 101100001101111010) it will be converted into the octal code which has 7 digits (As seen in the table 5144572 which is a code for E.Coli) (pls watch video for this AUTOMATED CULTURE METHODS BACTEC and BACT/ALERT are both automated microbial detection systems based on the carbon dioxide production of growing microorganisms. BACTEC uses fluorescent technology whilst, more on blood ● ● MALDI-TOF Proteomics using matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF-MS) has emerged as a reliable method for the rapid identification of microbial pathogens. This technology involves mixing of the pathogen with a suitable chemical matrix on a solid surface, pulsing the sample with a laser for ablation and desorption, followed by ionization using time-of-flight technology to create a mass spectrum of the samples. ○ This mass spectrum is then compared with a database of known spectra to allow for organism identification. ○ Fluid tube - vacuumed tube sa Maldi - Tof ms ○ Proteomics - study of proteins inside the cell WEEK 8: GRAM-POSITIVE COCCI (STAPHYLOCOCCUS SPP.) BALEROS. BELOCURA. CHEUNG. S, DEL ROSARIO. FORTUNO. FREKING. GAGUI. J, GARCIA. GUINTAPAN. LEAÑO. LOVERIA. MABUNGA. PACLEB | MT’25 | A.Y. 2023 - 2024 10/ 32