MT 6320: Clinical Bacteriology PDF

. MT 6320: CLINICAL BACTERIOLOGY Course Instructors:...

. MT 6320: CLINICAL BACTERIOLOGY Course Instructors: A.Y. 2024-2025, Term 1 ANTIMICROBIALS, ANTIMICROBIAL RESISTANCE AND AST 🗣️ Common Sites Affected: ○ Mouth ○ Respiratory tract ○ Genitourinary tract 🗣️ Ex: P. aeruginosa, C. albicans, and S. aureus 🗣️ NOTE: Wide-Spectrum Antibiotic Overuse: ○ It may lead to opportunistic infections of the Clostridium difficile. ○ May lead to seudomembranous colitis LEGEND ✎ Resistance would also happen if regimen of antibiotic is not followed. BOOK / OTHER FACTS & ARF LIVE ○ Ex. People would only take it for 1-2 days RESOURCES DETAILS TO NOTE DISCUSSION instead of 7 days ✄ ✎ 🗣️ SPECTRU ANTIBIOTIC ANTIMICROBIAL AGENTS M Definition: Substances that inhibit, kill or destroy PEP CBV microorganisms Narrow Sources: Spectrum Penicillin G ○ Microorganisms from bacteria or fungi 🗣️ More active against gram(+) bacteria ○ Chemically synthesized Erythromycin Types of Antimicrobial Agents: Polymyxin B ○ Antibiotics Clindamycin ○ Antibacterial agent Bacitracin, ○ Antiviral Vancomycin ○ Anti-fungal 🗣️ gram- positive ○ Anti-parasitic CAT Broad ACCORDING TO SPECTRUM OF ACTION Spectrum Cephalosporins NARROW SPECTRUM Ampicillin limited spectrum of action Tetracycline 🗣️ Can only target a certain group of microorganism ACTIONS OF ANTIMICROBIAL DRUGS BROAD SPECTRUM 1. Bactericidal agents - Kill or destroy the Wide spectrum of action microorganism ○ Action against Gr (+) & Gr (-) ○ Used in life-threatening infections and Disadvantage: Destruction/ Inhibition of the in infections in immunosuppressed normal flora leading to superinfections patients (since their immune system is not stable already) 1 2. Bacteriostatic agents - Inhibits the growth of microorganisms 🗣️ Does NOT directly kill or destroy the microorganism 🗣️ Given in patients who have a stable immune system (immunocompetent) 🗣️ Would still rely on the immune response of the patient. 🗣️ MAJORITY OF THE ANTIBIOTICS THAT WE Figure. Sites of Action of Antimicrobials USE INHIBITION OF CELL WALL SYNTHESIS BACTERICIDAL BACTERIOSTATIC 🗣️ Antimicrobials can target the cell walls and since cell walls are primarily linked to the bacteria. Aminoglycosides Chloramphenicol Antimicrobial class: Beta Lactams Beta lactams Quinupristin Mechanism of action Daptomycin Linezolid ○ Inhibit cell wall synthesis by binding Teicoplanin Erythromycin and enzymes involved in peptidoglycan Telavancin other macrolides production Penicillin-binding proteins [PBPs] Metronidazole Quinolones Trimethoprim Sulfonamides 🗣️ Substrate Blocking: ○ Levofloxacin Clindamycin Some antimicrobials also block Tetracycline substrate needed for cell-wall ○ Ciprofloxacin synthesis Rifampin Tigecycline Spectrum of Activity Vancomycin ○ Both Gram(+) and Gram(-) bacteria but Alpha Beta Delta TeTe Quick, LET’S Cry TT spectrum may vary with the individual MQRV (╥_╥) antibiotic ○ BETTER ACTIVITY AGAINST GRAM-POSITIVE BACTERIA 🗣️ Primarily used for the Gram (+) bacteria - The peptidoglycan layer is thicker and more exposed, making it more accessible 🗣️ Limited Activity against Gram(-)- thick peptidoglycan layer is less accessible ✎ MECHANISMS OF ACTION OF ANTIMICROBIALS Site of Action of the classes of Antibiotics [1 ] Microbial cell wall ​ Cytoplasmic Membrane ​ Chromosome function and replication ​ Protein synthesis Anabolic cellular processes targeted by Antimicrobial classes targeting cell wall synthesis antibiotics: ○ Beta-Lactam Antibiotics [A] Folate Synthesis Penicillins [B] DNA Replication Cephalosporins [C] RNA Transcription Monobactams [D] mRNA Translation Carbapenams ○ Glycopeptides ○ Cycloserine 2  B-LACTAM ANTIBIOTICS Penicillinase Resistant Penicillins Mechanism of action 🗣️ Only problem with this type of drugs is that ○ Inhibition of enzymes needed for there are already recorded cases of resistance peptidoglycan formation to these drugs. BETA-LACTAM COMBINATIONS 🗣️ To counteract this, we also have developed prodrugs such as the Piptaz, Augmentin, Composed of a beta-lactam drug with Sulbactam, Ampicillin. antimicrobial activity and a beta lactam drug Antimicrobials that have without activity components in order to minimize or ○ Ex. Ampicillin-sulbactam; prevent resistance amoxicillin-clavulanic acid; piperacillin-tazobactam ○ Effective against organisms that produce CEPHALOSPORINS beta lactamases that are bound by the Cephalosporins source: Acremonium inhibitor. Structure: ○ Similar to penicillin PENICILLINS ○ More resistant to beta-lactamases (enzymes Penicillin source: Penicillium notatum used to degrade the beta-lactam drug) and 🗣️Mechanism of Action: more modifiable ○ Beta-lactam drug that inhibits the Cephalosporin C - original cephalosporin enzyme for the peptidoglycan formation. ○ Modified to aminocephalosporanic acid - ○ Inhibits PBPs, also called the Easily modifiable transpeptidase. ○ Modified ot Further modified to 1st up to ○ Can form covalent complexes with 5th generation Cephalosporins elements like the PBPs which inhibit the 🗣️ Each generation would have varying cross-linking of the cell wall spectrum of action (narrow, broad, or Examples: expanded) Type Drugs GENERATION EXAMPLES SPECTRUM OF ACTIVITY Natural Penicillins Penicillin G 🗣️intravenously 1st Gen Cefaclor Narrow Spectrum Penicillin V 🗣️orally Cefazolin Penicillinase Resistant Nafcillin 2nd Gen Cefprozil Expanded Oxacillin Cefuroxime Spectrum Methicillin 3rd Gen Cefixime Broad Spectrum Extended Spectrum Aminopenicillins (AAA) Penicillin ○ Ampicillin Cefotaxime ○ Amoxicillin Ceftazidime Carboxypenicillin (CTT) ○ Carbenicillin, 4th Gen Cefepime Extended ○ Ticarcillin Spectrum Acycalmminopenicillins Ureidopenicillins 5th Gen Ceftaroline Broad, including Penicillin Co-drugs ​Augmentin Ceftobiprole MRSA coverage Pip-Taz Ampicillin/ Sulbactam Cephalosporin- Ceftolozane-tazobactam beta-lactamase Ceftazidime-avibactan ✎ Issue with Cephalosporins and Penicillins: inhibitor They develop resistance & produce combination beta-lactamases, thereby rendering your penicillins useless 3 MONOBACTAM ○ We do NOT give vancomycin or any other glycopeptides to Gram-negative Example: Aztreonam organisms or if you are considering ○ Narrow Spectrum gram-negative infections CARBAPENEMS ○ There are certain Gram-positive bacteria (Lactobacillus, Pediococcus) that may be Examples: (DIME) RESISTANT to vancomycin. ○ Doripenem ○ Certain Gram-negative bacteria ✄ Used for treatment of urinary tract & (Porphyromonas) can be SUSCEPTIBLE intraabdominal infections against vancomycin ✄Activity agaisnt: gram (-) and gram We have to monitor the therapeutic index when we (+) bacteria use vancomycin ○ Imipenem ○ Meropenem ✎ When there is resistance to penicillin ○ Ertapenem Glycopeptides are given when there is Broad Spectrum - similar to 3rd generation resistance to penicillin cephalosporins, slightly greater activity against ○ Targets the substrate Enterics (E. coli, Klebsiella sp.) Pseudomonas and ○ Vancomycin (good example) anaerobes Not effective for MRSA (Methicillin Resistant S. CYCLOSERINE aureus) and VRE (Vancomycin Resistant Enterococci) 🗣️​↓ Low activity against GRAM-POSITIVE bacteria MOA: inhibit the synthesis of the peptidoglycan 🗣️↑ More effective against GRAM-NEGATIVE bacteria precursors in the cytoplasm ○ 2nd line drug for TB OTHER DRUGS TARGETING CELL WALL SYNTHESIS INHIBITION OF CELL MEMBRANE FUNCTION GLYCOPEPTIDES (DISRUPTION OF CELL MEMBRANE) MOA: Binds to terminal D-Ala-D-Ala of the pentapeptide-glycosyl peptidoglycan POLYMYXINS intermediates Cyclic Polypeptides Examples: Examples: Polymyxin B and Polymyxin E ○ Vancomycin (Colistin) ○ Dalbavancin MOA: Act like detergents which interact with ○ Teicoplanin phospholipids, Increasing permeability (disrupt ○ Oritavancin the cell membrane) ○ Telavancin Clinical Use: Agent of LAST resort to P. aeruginosa TAKE NOTE: Activity is limited to Gram (+) and Acinetobacter infections (Gram(-) non-fermenting Organisms ONLY 🗣️ Clinical Spectrum: Primarily for Gram-positive bacteria) 🗣️Known for causing nosocomial or bacteria; aerobic clinical infections caused by: hospital-acquired infections which are ○ Staphylococci Toxicity: Neurotoxic and nephrotoxic ○ Streptococci ○ Enterococci 🗣️ Another downside is that this drug may 🗣️ DRUG OF CHOICE for MRSA: Vancomycin also act on the human cell membrane, 🗣️ LIMITATIONS: BACITRACIN not only the bacterial cell membrane. ○ Gram-negative organisms are inherently RESISTANT to Vancomycin. Source: Bacillus licheniformis due to the size of the molecule MOA: Inhibits the transport of lipid-bound of vancomycin, which precursors across the cytoplasmic membrane prevents passage through 🗣️ Activity against Gram-positive bacteria porin channels Toxicity: TOXIC; limited to topical application 🗣️ Not given orally since it is quite toxic 4 LIPOEPTIDES SULFONAMIDES Examples: Daptomycins Example: Sulfamethoxazole (SMZ) MOA: Disrupt membranes of Gr (+) bacteria MOA: Effective Against: ○ Competitive inhibition of folic acid ○ Vancomycin-resistant Staphylococcus aureus synthesis by binding to dihydropteroate (VRSA) synthase (analogue PABA) ○ VRE Moderately Toxicity: ○ MRSA ○ Vomiting, nausea, hypersensitivity reactions SUMMARY ○ Can be antagonistic to certain medications POLYMYXINS BACITRACIN LIPOPEPTIDES 🗣️ Warfarin (blood thinner) Ex: Polymyxin Bacitracin Daptomycin 🗣️ Phenytoin (neurologic diseases or B disorders) Polymyxin E (Colistin) 🗣️ oral hypoglycemic agents (diabetic) Clinical Spectrum: MOA Detergent-like action on Inhibits transport of Disrupts membranes 🗣️ Usually given for GRAM-NEGATIVE phospholipids lipid-bound of Gram(+) infections: Enterics (UTIs) , increasing precursors bacteria permeability across the and membrane TRIMETHOPRIM (TMP) disrupting the cell MOA: Blocks the step leading to formation membrane. tetrahydrofolate by preventing dihydrofolate reductase mediated recycling of folate coenzymes Clinical Last resort for Effective Treatment ✄ mediate formation of tetrahydrofolate (THF) Use Pseudomonas against for VRSA, aeruginosa Gram(+) VRE, MRSA from dihydrofolate and bacteria Effective in: Anaerobic environments Acinetobacter Clinical Spectrum: (Gram (-) non-fermenti ○ Anaerobes, ng bacteria) ○ Microaerophiles infections ○ Protozoans ○ Gardnerella Toxicity Neurotoxic, Highly Generally nephrotoxic; toxic; well-tolerate ○ C. difficile may affect limited to d but should Clinical Uses: human cell topical be 🗣️ Patients with Chronic UTI membranes application, not for oral monitored for muscle 🗣️ Enteric infections (E. coli, Haemophilus, use toxicity Moraxella) INTERFERENCES WITH DNA SYNTHESIS INHIBITION OF FOLATE SYNTHESIS Folate - important precursor in DNA synthesis of FLUOROQUINOLONES “QUINOLONES” bacteria Example: Nalidixic Acid ○ Enzymes mediating folate synthesis MOA: bind and interfere with Topoisomerase II Dihydropteroate synthase (DNA gyrase) & Topoisomerase IV (Sulfonamides) 🗣️ These two enzymes relieve the torsional Dihydrofolate reductase stress during the uncoiling of the DNA. (Trimethoprims) 🗣️ TARGETS: DNA GYRASE: Gram (-) Bacteria TOPOISOMERASE IV: Gram (+) Bacteria Toxicity: Tendinitis; rupture of Achilles tendon 5  Clinical Spectrum: Chlamydia ○ For Gram (-) bacteria 🗣️ Toxicity: Red discoloration of urine Neisseria Enterics INHIBITION OF PROTEIN SYNTHESIS Pseudomonads 🗣️ mRNA Translation: Inhibits Ribosomal Action Staphylococcus ○Ribosomes are necessary for protein Streptococcus synthesis and it is needed for translation to occur. METRONIDAZOLE Inhibits Ribosomal Action ○ Targeting 50S Ribosomal subunit (LRU) MOA: Nitro group is reduced by nitroreductase ○ Targeting 30S (SRU) leading to generation of cytotoxic compounds and free radicals that disrupt DNA that leads to INHIBITS 50S RIBOSOME bactericidal effect 🗣️ Free radicals- are quite toxic to the DNA CHLORAMPHENICOL and could lead to a bactericidal effect Effective in: Anaerobic environments MOA: inhibits translation through inhibition of 🗣️ In a reduced state elongation step; preventing attachment of AA Clinical Spectrum; For: 🗣️prevents elongation step or the ○ Anaerobes transpeptidation) Broad Spectrum ○ microaerophiles ○ protozoans 🗣️ Used for GRAM-POSITIVE, ○ Gardnerella GRAM-NEGATIVE, and ANAEROBIC BACTERIA (Bacteroides fragilis) ○ C. difficile 🗣️Clinical uses: used to treat typhoid fever (in the past) INTERFERENCES WITH RNA SYNTHESIS Toxicity: ✄ Transcription: DNA to RNA by RNA polymerase ○ Aplastic Anemia (pancytopenia) ✄ Bacterial RNA polymerase is a core tetramer ○ Gray Baby Syndrome (unable to metabolize composed of chloramphenicol) ○ 𝛂 subunit ○ 2 𝛃 subunit (𝛃 𝛃 ‘) MACROLIDES ○ 𝛄 subunit MOA: inhibits protein synthesis by binding to 23s ○ Dissociable 𝝳 subunit (controls RNA of the 50S ribosomal subunit inhibiting transcription of specific gene classes) transfer RNA ✄ allows initiation and mRNA translation to RIFAMYCINS (RIFAMPIN / RIFAMPICIN) begin but inhibits / block peptide MOA: binds to DNA dependent RNA polymerase to elongation inhibit RNA synthesis Examples: Clinical Spectrum: ○ Erythromycin ○ Primarily for Gram (+) Organisms; ○ Clarithromycin ○ 1st line TB treatment ○ Azithromycin 🗣️ Facultative intracellular bacteria ○ Clindamycin (Lincosamide) since it can be found inside ○ Erythromycin macrophages) 🗣️ Clinical Use: ✄ treat M. tuberculosis infection in treatment of acute pimples or topical combination w/ other antibacterial formations. classes DOC against Legionnaire’s disease (caused ○ Prophylaxis for N. meningitidis carriers by Legionella pneumophila): Erythromycin 🗣️ Intracellular pathogens (drug can be Also for the treatment of various intracellular concentrated within the cells) agents Rickettsia Mostly Bacteriostatic 6  Clinical Spectrum: INHIBITS 30S RIBOSOME ○ Intracellular pathogens (can penetrate TETRACYCLINE & DOXYCYCLINE WBCs ○ Gram(+) bacteria ✄ Members of polyketide chains of antibacterials, ○ Mycoplasma (cell-wall deficient represented by: Tetracycline, Doxycycline, bacteria), Minocycline ○ Treponemes (Treponema pallidum) MOA: Inhibits protein synthesis by binding ○ Rickettsia reversibly to the 30s ribosomal subunit Broad spectrum 🗣️ NOTE: ○ Bacteriostatic 🗣️Can chelate minerals (e.g., calcium and We do NOT give penicillin against mycoplasma magnesium), affecting bone growth and infections dental health. We do not give to much Clindamycin because it can ○ Also for intracellular pathogens, cause pseudomembranous colitis (associated with Mycoplasma(cell-wall deficient),, opportunistic bacteria such as Clostridium difficile) Spirochetes, Shigella Toxicity: OXAZOLIDINONES (synthetic) ○ GI irritation Examples: Linezolid ○ Phototoxic dermatitis ○ Hepatotoxicity (liver) ○ For Gram (+) Mycobacteria 🗣️ Nephrotoxicity (kidney) ✄ Binds to 50S ribosomal subunit, ○ Prevents formation of preinitiation 🗣️Contraindicated for pregnant women since complex with 30s subunit containing it may lead to discolored teeth and depressed initiation factors bone growth on born child ○ Blocks initiation step and translocation of peptidyl-tRNSA from A site to P site AMINOGLYCOSIDES ✄ Clinical Spectrum: gram(+) bacteria; MRSA, VRE, S. ✄ Cationic carbohydrate-containing molecules pneumoniae, M. tuberculosis ✄ MOA: ○ Interfere with initiation of bacterial ✄ STREPTOGRAMINS- QUINUPRISTIN-DALFOPRISTIN ribosome MOA: ○ Mistranslation / misreading of codons, ○ Interferes w/ elongation of polypeptide producing aberrant proteins chain ○ Incorporation of aberrant proteins into cell ○ Prevents binding of aminoacyl-tRNA to the wall enhance cellular penetration to ribosome and formation of peptide bonds bacterial inner membrane ○ Stimulates dissociation of peptidyl-tRNA, ○ Penetration to bacterial inner membrane and interferes with the release of requires aerobic environement for polypeptide by blocking the exit tunnel activaiton; this is why aminoglycosides where it leaves the ribosome lacks activity against anaerobic bacteria Combination of two streptogramins with a 70:30 Often used together with penicillins (for better ratio activity) in order to diffuse and enter bacterial cell ○ Streptogramin A - Dalfoprisitn (70%) Examples: ○ Streptogramin B - Quinupristin (30%) ○ Streptomycin Dalfopristin & Quinuprisitn are synergistic ○ Gentamicin because of enhance affinity of quinuprisitn for the ○ Tobramycin ribosome ○ Amikacin ○ Neomycin Toxicity: Ototoxic, nephrotoxic ○ Narrow Therapeutic Index 🗣️ The dose of the drug is very narrow for it to have a toxic and effective dose 7  🗣️ Too MUCH = toxicity 🗣️ 🗣️ Too LOW = ineffective Line 3rd Linezolid Inhibits protein synthesis by binding to the 50S ribosomal ○ We have to monitor the trough and peak Drugs subunit. levels to avoid toxicity Macrolides Inhibit protein synthesis; ○ Trough levels: blood sample within 15-30 effective against some mins before the next dose mycobacterial infections. ○ Peak levels: obtain blood sample within 30 mins (IV) or 60 mins (IM) following the Rifabutin Similar action to Rifampicin; used for resistant strains. most recent infusion of antibiotic ○ Other drugs that have narrow therapeutic index include the Vancomycin and MECHANISMS OF ANTIBIOTIC RESISTANCE Chloramphenicol BIOLOGICAL RESISTANCE ✄ GLYCYLCYLINES - TIGECYCLINE Changes that result in observably reduced Derivatives of the tetracycline class susceptibility of an organism to a particular antimicrobial agent Carry a glycylamido moiety attached to 9-position of minocycline 🗣️ Antibiotic is still working but already at a reduced MOA: Inhibits protein synthesis by binding susceptibility which needs a higher dose for it to reversibly to 30s ribosomal subunit work ○ Blocks entry of aminoacyl-tRNA molecules CLINICAL RESISTANCE onto A site of ribosome Antimicrobial susceptibility has been lost ○ Preventing incorporation of AA residues Drug is no longer effective for clinical use into elongating peptide chains 🗣️ In the past, antibiotic is still working, but lost ○ Has stronger binding affinity to 30s susceptibility through the years ribosomal subunit than tetracycline 🗣️ Ex: Penicillin was used before as a drug for ○ Has more potent activity against gonorrhea, but penicillin lost its activity against it. tetracycline-resistant- organisms with There were already reported cases of resistance efflux and ribosomal protection against the said drug mechanisms of resistance ENVIRONMENTALLY MEDIATED ANTIMICROBIAL Broad Spectrum: gram (+) & gram(-) bacteria, RESISTANCE some mycobacteria and anaerobic pathogens 🗣️ This includes the conditions where the bacteria and the antibiotic is observed in the performance of ANTIMYCOBACTERIAL AGENTS susceptibility testing 🗣️ You have to standardized the certain Drug Drug Name Mechanism of Action environmental factors Line 🗣️ Includes pH ○ There are certain antibiotics that would 1st Line Isoniazid Interferes with the formation of Drugs mycolic acid. have false resistance or decreased susceptibility if the pH of the medium is Rifampicin Inhibits DNA-dependent RNA becoming more acidic polymerase. ENVIRONMENTAL FACTOR Pyrazinamide Bactericidal. pH Decrease Leads to aminoglycoside 2nd Line Ethambutol Inhibits mycolic acid d pH and erythromycin Drugs formation. ( ACQUIRED MECHANISMS OF RESISTANCE 🗣️ Definition: Resistance that develops due to changes or alterations in the physiology, structure, or genetic makeup of bacteria. It can occur unpredictably and may involve the transfer of resistance genes. 🗣️Key Characteristics: ○ Physiological Changes: Alterations in how bacteria function can lead to resistance. ○ Genetic Changes: Mutations or gene acquisition can create resistance profiles. ○ Horizontal Gene Transfer: Resistance can spread through mechanisms like conjugation, transformation, or transduction Specific genes Associated with Acquired Resistance ○ Efflux Mechanism: Changes in the genes BIOFILMS coding for the efflux pump mefA gene: in Streptococcus 🗣️ Groups of bacteria that form a matrix (protective pneumoniae leading to macrolide cover) preventing them from being targeted by the resistance antimicrobial msrA gene - S. aureus and Sessile bacterial communities Enterococcus against macrolides Irreversibly attached to solid surfaces mreA gene - S. Agalactiae against Embedded in exopolysaccharide matrix macrolides Leads to decreased penetration of antibiotics and the formation of persister cells ✄ Resistance: MECHANISMS OF ACQUIRED RESISTANCE ○ Bacteria in biofilms are highly Occurs as a result of prior exposure resistant to antimicrobial agents and Caused by changes in the genetic make up host-defenses 🗣️ May involve the transfer of resistance ○ Resistance is influenced by chemical genes via conjugation & physical characteristics of biofilm Unpredictable formation Genetically Encoded via: ○ Successful genetic mutation ○ Acquisition of genes via gene transfer ○ Combination of mutational and gene transfer 🗣️ Key Example: mecA Gene 10  Note: NOT all organisms are subject to ST ✄ RESISTANCE TYPES ○ Examples: S. pyogenes, S. galactiae, N. INNATE RESISTANCE INDUCED RESISTANCE meningitidis, L. monocytogenes Activated as part of From induction by biofilm developmental antimicrobial agent itself pathway 🗣️ Decreased permeable penetration of antibiotics 🗣️ Results in formation of Resulting in differential persister cells (parang resistance gene makulit na bacteria na expression throughout hindi mapatay-patay) biofilm community AST (ANTIMICROBIAL SUSCEPTIBILITY TEST) Purpose: Performed on clinically significant REASONS AS TO WHY AST IS PERFORMED bacteria Provides information on the decrease of the 🗣️ This is the organism that is being antimicrobial susceptibility associated with the patient 🗣️ AST CRITERIA DETAILS The next step of the doctor is to know what the appropriate antibiotic or antimicrobial Performed on Bacterial isolates that are the is given probable cause of infection 🗣️ The doctor will ask the laboratory what will be the appropriate antibiotic/s that can NOT performed Bacterial isolates that are work for the patient. on predictably susceptible to a 🗣️Guidelines for Performing AST: certain agent ○ Only on Clinically Significant Isolates ○ AST should NOT be performed on normal FACTORS TO CONSIDER IF AST IS flora. NEEDED/WARRANTED ○ Exceptions: Certain clinically significant organisms (e.g., S. pyogenes, S. galactiae, N. FACTOR CONSIDERATIONS meningitidis, L. monocytogenes) do NOT require AST due to well-known Body Site Do NOT perform AST on bacteria susceptibility profiles. isolated from its natural habitat Primary Goal: ✎ stool: E.coli is normal flora of ○ To provide STANDARDIZED in-vitro testing the GI tract so normally AST is not of a bacterial pathogen to a set of available performed antibiotics to determine its “antibiogram” ✎ throat culture: V. streptococci in order to predict the in vivo effectiveness is normal of particular antibiotic or antibiotic regimen Presence of Isolation from a pure culture is less 🗣️ Importance of Antibiograms other bacteria & likely to be contaminated ○ Used to compare the susceptibility of Presence of >2 species at >105 CFU quality of particular bacterial isolates to different (colony forming units) /ml in urine is Specimens antibiotics possibly due to contamination ○ Helps identify if bacteria are developing resistance to certain antimicrobials. Host Status Normal Flora might be the cause of ○ Important to the doctor to investigate any the infection in cases of unusual resistance patterns immunocompromised patients. Note: ONLY Implicate/ probable bacteria causing the infection should be tested! 11  Incubation temperature ✄ Patients who are allergic to Incubation duration penicillin and have a B-hemolytic Antimicrobial concentrations streptococcal infection, drug of Guidelines & Standards choice are erythromycin / ○ CLSI: updates and publishes standards clindamycin 🗣️ EUCAST (European Committee on INOCULUM STANDARDIZATION OF AST Antimicrobial on Antimicrobial Standardized Inoculum must be used within 15 Susceptibility Testing): agency where we minutes can follow microbiology protocols 🗣️ You have to immediately inoculate it in the ✄ ISO (International Organization for plate because doing it beyond 15 minutes Standardization): guides WORLDWIDE can already affect the result of the AST. antimicrobial susceptibitliy testing; a Comparison is done visually worldwide federation of national Nephelometric or spectrophotometric methods standard bodies can be done for more precision ○ At 625 nm, the absorbance should be INOCULUM STANDARDIZATION PREPARATION ranging from 0.08 - 0.1 1. Picking of colonies: ○ Use 4-5 similar-looking colonies from a non-inhibitory medium (NA or TSA) 🗣️ If fastidious, pick from an enriched media. 2. Suspension preparation: ○ Transfer picked colonies to broth medium (Nutrient Broth or Trypticase Broth) and grow to log phase (3-5 hours). Three important Purposes: 1. To optimize bacterial growth conditions to ensure that the inhibition of growth can be attributed to the antimicrobial agent. 2. To optimize conditions for maintaining antimicrobial integrity and activity, attributing the failure to inhibit bacterial ALTERNATIVE: growth to organism-associated resistance. Pick 4-5 colonies (Fastidious ​Organisms) from a 3. To maintain reproducibility and consistency fresh (16-24hr) culture and suspend in broth or in the resistance profile of an organism, NSS diluted to proper density regardless of what laboratory performs the NO need to incubate test. 3. After incubation, the turbidity of the broth is STANDARDIZED COMPONENTS standardized by comparing it with the 0.5 Bacterial inoculum size McFarland Standard Growth medium ○ pH ○ Cation concentration ○ Blood and serum supplements ○ Thymidine content Incubation atmosphere 12  MCFARLAND TURBIDITY STANDARD METHODS OF AST 🗣️ Purpose: Directly measure the activity of one or more ○ Used to standardize the turbidity of antimicrobial agents bacterial inoculum for antimicrobial Directly detect the presence of a specific resistance susceptibility testing. mechanism Preparation: Special methods that measure complex ○ Commercially available and widely used antimicrobial-organism interactions standard (can be prepared in-house) Methods that directly measure the activicity of one ○ 0.5 McFarland Standard or more antimicrobial agents: 99.5 ml 1% Sulfuric Acid + 1. TRADITIONAL AST METHODS 0.5 ml 1.175% Barium chloride a. Disk Diffusion 🗣️ The Wickerham Card is used to help in the 🗣️ ​Does not give the exact standardization process (helps in concentration that will inhibit determining proper turbidity) the organism (MIC - Minimum Characteristics: Inhibitory Concentration) ○ Corresponds to 1.5 X 108 CFU/ml b. Quantitative Dilution Susceptibility 🗣️ Specimen should be very similarly looking Testing - determination of MIC to your McFarland standard. ​Methods: 🗣️ Kung gaano kalabo yung McFarland Agar Dilution standard, dapat ganun din kalabo yung Broth Dilution (Micro inoculum and Macro) 🗣️May adjust the turbidity by the sterile broth or sterile NSS (if too turbid)- continuously 🗣️ Usually expressed in mg/mL adding NSS or broth to the inoculum, 🗣️ LOWER MIC or breakpoint = MORE eventually, the turbidity will be very similar EEFFECTIVE the antibiotic. to the McFArland standard 🗣️ Increasing MIC = suggests that it 🗣️ If it is less turbid, you put it back in the requires MORE antibiotic or may incubator to make it more turbid be developing a resistance 🗣️ Testing Conditions: pattern already ○ The best time for testing the activity of 2. COMMERCIALLY AVAILABLE METHODS antimicrobials is if the organisms are in 3. SPECIAL SCREENS & INDICATOR TESTS their logarithmic states. 🗣️ Storage: must be at a room temperature and kept 1. TRADITIONAL AST METHODS DISK DIFFUSION METHOD - KIRBY BAUER in the dark (in an amber bottle) ○ Usually, it may last for 6 months 🗣️ Recommended method for Aerobic, fastidious organisms Quality Control: McFarland Standard should be Commonly used in vitro tests in the laboratory checked monthly Inexpensive, easy to perform ○ At 625 nm, the absorbance should be Procedure: ranging from 0.08 - 0.1 ○ Inoculating a standardized bacterial suspension on Mueller Hinton Agar (MHA) CHOICE OF ANTIMICROBIALS ○ Antibiotic Disks are then placed, then Selection of Test Batteries/Panel (usually 10-15 incubated antimicrobials) depending on: ○ Antibiotic diffuses into the medium and a ○ Protocol of Hospital zone of inhibition is formed ○ ID of the organism (if significant, normal or Measurement: contaminant) ○ Diameters of zones of inhibition (ZOI) are ○ Any known resistance patterns measured in mm ○ Method of AST ○ Reported as: ○ Availability of Antimicrobial Agents 13 SUSCEPTIBLE 🗣️ Appropriate choice of antibiotic INCUBATION INTERMEDIAT 🗣️LESS effective compared to a 35℃ for 16-18 hours at ambient atmosphere (no CO2) E susceptible result 🗣️ CO2 can affect the antibiotic in a way that it RESISTANT 🗣️NOT recommended or not makes the MHA agar more acidic which leads appropriate to decreased activity of certain antibiotics Fastidious Organisms: 5-10% CO2 KIRBY BAUER METHOD PRINCIPLE: READING PLATES Gradient of antimicrobial agent is formed Examine plates if it grown satisfactorily The manner of streaking in a Mueller Hinton Agar Must be confluent is what we call the Overlapping Streak Method 🗣️ You have to rotate the plate and completely ○ Homogeneous growth and no isolated colonies cover all of the surface of the plate 🗣️ It is done for us to have a confluent growth No individual colonies should be present!! Specific considerations for certain organisms: As the agent diffuses farther, the concentration decreases until it reaches a point when the ○ For Proteus: hazy/ swarming growth within the bacterial growth is not anymore inhibited zone should be ignored during reading ○ For testing sulfonamides and TMP: hazing ZONE OF INHIBITION should also be ignored ○ Beta-hemolytic bacteria: ignore the 🗣️ Measured using a ruler or vernier caliper hemolysis produced expressed in mm 🗣️ MRSA: use of transmitted Light ○ Reflected light is also used for better 🗣️ Staphylococcus testing: Oxacillin visualization of the ZOI 🗣️ Enterococcus: Vancomycin Based on the inverse linear relationship between the zone of inhibition diameter and the logarithm of the minimum inhibitory concentration (MIC) 🗣️ The larger the zone of inhibition, the MORE susceptible, the lower the MIC Standards and Protocol are based on the CLSI Guidelines ✄ Figure. Escherichia coli tested by the disk diffusion method. The lawn of growth following overnight incubation shows individual colonies, representing unsatisfactory growth. The most likely explanation for the scanty growth is the use of an inoculum that is too light or contains too many nonviable cells, resulting in larger than normal zones and potentially false-susceptible results. 🗣️ PLATE SPECIFICATIONS 100 mm Plate: Up to 5 antibiotic disks. 150 mm Plate: Up to 12 antibiotic disks. Spacing Requirements ✄ Figure. Klebsiella (Enterobacter) aerogenes tested by the disk diffusion ○ Distance from the edge: 10-15 mm method. Zone measurements confirm that the isolate is susceptible to all ○ Distance between disks: at least 24 mm. 🗣️ agents tested. We have to follow this to avoid the formation of EXCEPT ampicillin (at the 1-o'clock position) and cefazolin (at the overlapping zones of inhibition 2-o'clock position). No zones are present for either of these agents. 14  ○ Susceptible- Inhibited by achievable concentrations in vivo. ○ Intermediate - Approaches usual levels, lower response rates ○ Resistant - Not inhibited by achievable concentrations BROTH DILUTION Macrodilution & Microdilution 🗣️ For anaerobic organisms 3 general interpretations: S, I, R NB: Recently added categories include ○ Susceptible Dose Dependent ○ Non susceptible In some cases, Breakpoints are only reported ○ Breakpoints - specific concentrations that separate or define the different categories ✄ Organisms with MICs at or below breakpoint: Susceptible ✄ MICs above breakpoint: Intermediate / Resistant ✄ BREAKPOINT Susceptible Two conc. are tested = NO GROWTH present in either well 🗣️ MUTANT COLONIES Intermediate √ WITH growth in LOW conc. X NO growth in HIGH conc. Presence of colonies within ZOI may indicate developing resistance. Resistant √ GROWTH in BOTH wells Further testing is required. ○ We test the colonies and subculture them to perform another AST to determine the proper antibiotic ADVANTAGES & DISADVANTAGES OF KIRBY BAUER TEST Figure. In this case, the MIC is at test tube number 3 (from the right) 🗣️ Take note of the tube that has no growth Advantages: Convenient and User friendly 🗣️ The amount of antibiotic corresponds to the MIC Disadvantages: Not all organisms have interpretative criteria (cut-off value); Unable to 🗣️ The MIC value will have more significance if we know the cut-off value (whether it is already susceptible provide more precise data about the level of or resistant based on CLSI guidelines) resistance DILUTION SUSCEPTIBILITY TESTING Determine Minimum Inhibitory Concentration (MIC)- Lowest concentration of an antimicrobial agent in agar that completely inhibits visible growth Use of serial 2-fold dilutions of the antimicrobial agent (expressed as ug or mcg/ml) Once MIC is determined, it is interpreted as S,I,R 15  Feature Broth Broth Microdilution SUMMARY OF BROTH DILUTION SUSCEPTIBILITY Macrodilution Tests Tests TESTING CONDITIONS Method Use of test tubes Use of wells Volume 1-2 ml per tube 0.05-0.1 ml per well ✄ ADDITIONAL INFO Number of Test ONE type of Test MULTIPLE Antibiotics antibiotic only antibiotics and multiple organisms → (Impractical as a →more cost-effective routine method when several antimicrobial agents must be tested) Broth Mueller-Hinton Medium Broth for non-fastidious bacteria Standardize Final concentration Each well has d Inoculum of 5 x 10^5 CFU/mL standardized added to each inoculum dilution AGAR DILUTION TESTS Controls Growth control tube Growth control and Method: and uninoculated uninoculated control control tube used wells included ○ Antimicrobial Concentrations and Growth control tube organisms are placed together on an agar (broth + inoculum) & medium ( spot inoculation) Uninoculated control ○ Series of dilutions, with one dilution per tube (broth only) are plate used w/ each assay as a sterility check 🗣️ MIC is determined on the first plate showing no growth ✄ Shelf life: 1 week stored at 2° - 8℃ 🗣️ Incubation Overnight at 35°C Overnight at 35°C, Conditions tray placed on a Testing Capacity: reading device ○ You may test for different isolates or MIC Absence of turbidity Lowest concentration organisms on a single plate with one Determinati indicates the lowest with no obvious concentration of antibiotic only (up to 32 on concentration that growth is MIC different isolates per plate) inhibits growth ○ A SINGLE plate contains a single antibiotic Breakpoint Susceptible: NO Standard Inoculum: 1 x 10⁴ CFU/spot Interpretati growth in both Advantage: on concentrations ○ Reference Method for ANAEROBIC Intermediate: growth ORGANISMS in low concentration, no growth in high ○ Useful for determining the MIC of Resistant: growth in N. gonorrhoeae both wells 🗣️ H. pylori MBC MBC determined by Determinati subculturing negative on tubes onto BAP; first plate showing no growth indicates MBC Figure. The Steer’s replicator 16  E-TEST (EPSILOMETER TEST) SUMMARY OF AGAR DILUTION SUSCEPTIBILITY TESTING 🗣️ CONDITIONS Gradient Diffusion Susceptibility Test 🗣️ Also provide an MIC value 🗣️ A commercially available method 🗣️ If more than 1 day, may lead to false susceptibility Less inoculum, may lead to false susceptibility Principle: Establishment of an antimicrobial density gradient ○ Uses thin plastic strips with gradations ○ Placed in a radial fashion on an inoculated plate. Interpretation: MIC is read wherein the growth ellipse intersects the E-test strip Useful for fastidious organisms S. pneumoniae H. influenzae Other streptococci 🗣️ Anaerobic bacteria Plate Specifications ○ 150 mm plate: Up to 5 ○ 100 mm plates: Up to 2 only METHODS OF DETECTING ANTIBIOTIC RESISTANCE CHROMOGENIC BETA LACTAMASE TEST CEPHALOSPORINASE TEST (CEFINASE TEST) 🗣️Info provided by arf Target: presence of Beta-Lactamase Organisms: Usually produced by Staphylococcus app., N. gonorrhoeae, and Moraxella catarrhalis, etc. Reagent: Nitrocefin (inside the filter paper disk) ○ Chromogenic cephalosporin ○ Very sensitive ○ It has a color reaction ○ (+): deep red or pink Usually appear in 10 minutes from 2. COMMERCIALLY AVAILABLE METHODS the time of specimen application Diffusion in Agar Derivation If Staphylococcus, 60 mins E-test ○ (-): yellow 17  DETECTION OF MRSA/ OXACILLIN RESISTANT S. IN ENTEROCOCCI AUREUS Resistance Mechanisms (vanA & vanB= most OXICILLIN SCREEN AGAR common): 🗣️ Composition: MHA with 6ug/mL of oxacillin with ○ VanA gene (Van-A phenotype); 4% NaCl 🗣️Gene responsible for resistance CEFOTOXIN DISK SCREEN TEST 🗣️Inducible (high level resistance) ○ Van-B phenotypes Dosage: 30ug of cefoxitin Testing: ○ Vancomycin Screen Agar (6ug 🗣️ Cefoxitin is used because it has been found vancomycin BHI agar) out that it is an effective inducer of mecA resistance gene PENICILLIN RESISTANCE IN S. PNEUMONIAE 🗣️ Recommended screening for MRSA according to Testing: CLSI ○ MHA with sheep blood; 1ug/ml oxacillin CHROMOGENIC AGAR FOR MRSA disk (incubate with CO2) 🗣️ The medium has added cefoxitin ○ Interpretation: 🗣️ Results: Susceptible (S) if ≥ 20mm Resistant (R) or (i) if < 20mm ○ Positive: Mauve/rose color. HLAR (HIGH LEVEL AMINOGLYCOSIDE RESISTANCE) ○ Negative: Non-mauve/non-rose color Enterococci - known for intrinsic resistance to aminoglycosides; combination therapy with MOLECULAR METHODS vancomycin is better DETECTION OF mecA Gene and its products (PBP2a) Testing: 🗣️ GOLD STANDARD for identifying MRSA ○ BHI Agar- test inoculum with 120ug/ ml gentamicin and 300 ug/ ml streptomycin disk VANCOMYCIN RESISTANCE (S) if >10mm IN STAPHYLOCOCCUS (R) if