Lecture 2 - Microbial Growth and Control PDF

BIOL 2010 Lecture 2 – Microbial Growth and Control BAILEY AND SCOT T’S – CHAPTER 10 - 11 DIAGNOSTIC MICROBIOLOGY – CHAPTER 12, 13 In this lecture Describe the most commonly encountered methods of disinfection and sterilization utilized in the clinical microbiology laboratory Describe, with parti...

BIOL 2010 Lecture 2 – Microbial Growth and Control BAILEY AND SCOT T’S – CHAPTER 10 - 11 DIAGNOSTIC MICROBIOLOGY – CHAPTER 12, 13 In this lecture Describe the most commonly encountered methods of disinfection and sterilization utilized in the clinical microbiology laboratory Describe, with particular emphasis on mode of action and efficacy, frequently encountered antimicrobial agents utilized in the clinical setting Describe antimicrobial sensitivity testing and its importance in the clinical microbiology settings Agar dilution methods, Broth Dilution methods Modifications to routine AST methods Common supplementary testing D test B-lactamase test Screening tests for MRSA, VRE, ESBL, CPE Disinfection vs. Sterilization Sterilization is the destruction of all forms of life. All or nothing process Disinfection is elimination of a defined scope of microorganisms. Disinfectant: Chemical agents applied to inanimate objects Antiseptic: A substance applied to the skin to eliminate or reduce the number of bacteria present Types of Microorganisms and Resistance to Killing 4 Methods of disinfection/sterilization PHYSICAL METHODS CHEMICAL METHODS Heat: dry or moist Alcohols (50-70%) Including boiling, pasteurization Aldehydes Filtration Halogens Radiation Heavy Metals Lyophilization Detergents UV Phenolics Gases The Autoclave ** Typical Specifications Pressure: 15 psi Temperature: 121C Time: 15-20 minutes Autoclaving at a pressure of 15 psi at 121C for 15-20 minutes destroys vegetative microorganisms, bacterial endospores, and viruses Chemical Agents Commonly Used as Disinfectants and Antiseptics 7 Antimicrobial agents Organisms are eradicated by being actively inhibited or killed by a substance Antimicrobial agents are a group of natural and synthesized substances that target organisms Antibiotics are obtained and purified from other microbial organisms Antimicrobial agents that inhibit/halt bacterial growth are known as bacteriostatic Agents that usually kill the organism are bactericidal Antimicrobial mode of action Cell Wall DNA and Cell RNA Membran e TARG Synthesi s ET Protein Metaboli Synthesi c s Pathways Antimicrobial mode of action 10 11 Inhibition of Bacterial Cell Wall Biosynthesis Cell wall protects bacteria. Specific integral enzymes are necessary to build and shape the cell wall. Transpeptidases: cross-link the cell wall These enzymes are also called penicillin binding proteins (PBPs). Specific to each bacteria; therefore drugs may have difference in binding to each PBP and thus have different levels of effectiveness Drugs are designed to inhibit and inactivate these enzymes. 12 The Cell Envelope Structure of a Gram-Positive (Left) and Gram-Negative (Right) Bacterium 13 B Lactam antibiotics Bactericidal β-Lactam antimicrobial agents contain a four- membered, nitrogen-containing ring at the core β-Lactam antimicrobial agents bind the enzyme inhibiting transpeptidation and inhibit cell wall synthesis Note: β-lactams must pass through cell wall porins in gram-negative cells to reach cell PBPs. Based on chemical structure, the β-lactam ring Binds to and inhibits the transpeptidases (known as penicillin-binding proteins, PBPs) B Lactam antibiotics β-lactam antibiotics can be Natural and semi-synthetic Classes: Penicillins, cephalosporins, carbapenems, monobactams B Lactam antibiotics Spectrum of activity varies by type of drug Glycopeptides Bactericidal Bind to the end of the peptidoglycan, interfering with transpeptidation Inhibit cell wall synthesis and growth Large molecules and cannot penetrate gram-negative cell walls or parts of human body (ex. Blood-brain barrier) Many toxic side effects Examples: Vancomycin Spectrum of activity: Narrow spectrum (Gram Pos only) Used for Staphylococcus, Streptococcus, Enterococcus Cell Membrane Inhibitors -Polymyxin Bactericidal Usually act similar to detergents that interact with phospholipids to increase permeability Macromolecules and ions leak across the membrane to cause cell death Examples: Polymyxin B Colistin Spectrum of activity Are more effective against gram-negative bacteria Polymyxin antibiotics may be used as an agent of last resort for Pseudomonas aeruginosa and Acinetobacter spp. Protein Synthesis Inhibitors Target protein synthesis and severely disrupt cellular metabolism Bind 30s or 50s ribosomal subunits Types: Aminoglycosides MLS group (Macrolide-lincosamide-streptogramin group) Ketolides Oxazolidinones Streptogramin Chloramphenicol Tetracyclines Tigecycline Glycylglycines Protein Synthesis Inhibitors Aminoglycosides Bactericidal Irreversibly binds to 30S subunit Prevents docking of aminoacyl-tRNA Also contributes to misreading the genetic code Large molecule – cannot cross blood-brain barrier and cannot treat infections related to CNS (Ex. Meningitis) Side effects: nephrotoxic and auditory or vestibular toxicity Levels must be monitored Examples: Gentamicin, Tobramycin, Streptomycin, Kanamycin Spectrum of activity: Primarily Gram Negative (sometimes Staphylococcus aureus) Protein Synthesis Inhibitors Aminoglycosides Activity of Aminoglycoside depends on Ca and Mg cations Both cations and Aminoglycosdes both positively charged and compete for the negatively charged bacterial cell wall If there is too much Ca or Mg ion (Ex. In vitro testing, it will outcompete the drug) Therefore, INCREASED concentration of cations = LOWER effect of aminoglycoside Protein Synthesis Inhibitors Chloramphenicol Bactericidal Irreversibly binds to 50S subunit Prevents docking of aminoacyl-tRNA Side effect: Bone marrow toxicity (can cause aplastic anemia) Clinical use is VERY limited as a result When used – monitored with a CBC test Spectrum of activity: Broad spectrum - (Gram pos and Negative) including atypical pathogens like Chlamydia spp., Rickettsia spp., Coxiella burnetii, and Mycoplasma and some anerobes Protein Synthesis Inhibitors Glycylcycline Higher affinity for 30S ribosomal subunit than tetracycline Increased effectiveness against tetracycline-resistant organisms Side effects: Associated with side effects and increased mortality Example: Tigecycline Spectrum of activity Wide spectrum of activity against gram-positive, gram-negative, atypical, and anaerobic pathogens Used to treat complicated intra-abdominal infections, skin infections, and community-associated pneumonia  Protein Synthesis Inhibitors Macrolide-Lincosamide-Streptogramin (MLS) Group All protein synthesis inhibitors – mostly effective against gram positive, some gram negative Bacteriostatic or bactericidal (depending on concentration) Macrolide Lincosamide Streptogramin Spectrum of Most aerobic and anaerobic gram- Mostly Gram pos, some gram Primarily Gram Positive Bacteria activity positive bacteria and atypical neg, bacteria Clindamycin primarily for anaerobes Examples Erythromycin, Azithromycin, Clindamycin Quinupristin/dalfopristin – Clarithromycin synergistic or combination drug Notes Most commonly used antibiotic in the Clindamycin-associated MLS group diarrhea possible. Leads to secondary infections by C. difificile. Protein Synthesis Inhibitors Oxazolidinones Binding 50S subunit and blocking initiation and translocation Side effects: Long term therapy associated with risk for serious hematologic and neurologic toxicity Examples: Linezolid Spectrum of activity: Narrow spectrum – mostly used for MRSA, VRE and Mycobacterium tuberculosis Protein Synthesis Inhibitors Tetracyclines Bacteriostatic Reversible binding to 30S subunit Inhibit rotation of bound tRNA into the A site Causes premature release of peptides Examples: Tetracycline, doxycycline, minocycline Spectrum of activity Broad Spectrum including atypical bacteria such as: Mycoplasma, Intracellular pathogens (Treponema pallidum, Chlamydia, Rickettsia), Spiral bacterium ex. Borrelia, Vibrio cholerae Inhibitors of DNA/RNA Synthesis Metronidazole Bactericidal A type of nitroimidazole antibiotic Nitro group is reduced in bacterial cytoplasm, generating cytotoxic compounds that disrupt DNA Activation of metronidazole requires reduction under conditions of low redox potential, such as anaerobic environments Spectrum of activity: Used primarily for Anaerobic infections, Can be used for some protozoans (Trichomonas, Giardia, Entamoeba) Inhibitors of DNA/RNA Synthesis Quinolones Original quinolone: Nalidixic acid (narrow spectrum) Fluoroquinolones have enhanced activity Bind to and interfere with DNA gyrase enzymes. Newer quinolones inhibit topoisomerase IV (similar to DNA gyrase) Side effects: Associated with many toxic effects ex. aortic dissections, tendinitis Examples: Ciprofloxacin, Levofloxacin, Moxifloxacin Spectrum of activity: Broad Spectrum Uses in treatment of Enterobacteriaceae, Pseudomonas, Staphylococci, Enterococci, Neisseria, and Streptococci species other than S. pneumoniae Inhibitors of DNA/RNA Synthesis Rifamycin Binds to DNA-dependent RNA polymerase and inhibits RNA synthesis Example: Rifampin is a synthetic derivative of rifamycin B, targets DNA transcription Structure doesn’t allow penetration on most gram negative cell membranes Spectrum of activity Narrow spectrum (Gram Pos only) Used in synergistic therapy - in combination with other antibacterial classes to treat Mycobacterium tuberculosis. Metabolic Inhibitors Folic acid is essential for bacterial DNA – most bacteria make their own folic acid Folic acid synthesis mediated by two enzymes: Dihydropteroate synthase and Dihydrofolate reductase Sulfonamide Inhibits dihydropteroate synthase in the folic acid pathway Needs constant levels of drug to inhibit enzyme Trimethoprim Inhibits dihydrofolate reductase (DHFR) in the folic acid pathway Needs constant levels of drug to inhibit enzyme Metabolic Inhibitors Using Sulfonamide and Trimethoprim in combination offers a synergistic effect (SXT) Bactram and Septra – common names Synergy Combined effect is greater than the additive effect. Enhanced activity Spectrum of activity of folate pathway inhibitors Broad spectrum - provides activity against the Enterobacteriaceae that cause urinary tract infections Metabolic Inhibitors Increased concentrations of thymine (Ex in vitro, in media) will allow bacteria to circumvent the effects of folic acid inhibitors Therefore greater thymine concentrations will negate the use of folic acid inhibitors Antimicrobial resistance mechanisms INTRINSIC RESISTANCE ACQUIRED RESISTANCE Naturally (innate) found in Acquired from exogenous DNA bacteria (chromosomal) (plasmid, conjugation, Transmitted to progeny vertically transposons, bacteriophage, etc.) Big concern in health-care Predictable once the organism is settings identified Example: ESBL Example: Gram negative bacteria are resistant to Vancomycin Examples of Intrinsic resistance Antimicrobial resistance mechanisms Possible causes for antimicrobial resistance Lack of affinity of the drug for the bacterial target Impermeability: Inability of the drug to enter the bacterial cell Removal of the drug by chromosomally encoded efflux pumps Innate production of enzymes that inactivate the drug Biofilms Antimicrobial resistance mechanisms Enzymatic inactivation of antimicrobials Some bacteria produce enzymes that destroy the drug before they are able to reach their targets Example β-lactamases = most COMMON method of resistance to beta lactam antimicrobial Variety of B lactams β-Lactamase Inhibitors β-Lactamase Inhibitors Share structure similar to β-lactam antimicrobials. Have little to no antimicrobial activity. Act to bind β-lactamase enzymes and allow other β-lactam agent in the combination to exert its antimicrobial activity. Examples: Sulbactam, clavulanate, tazobactam, avibactam Examples of β-lactam/β-lactamase inhibitors combinations Ampicillin/sulbactam and amoxicillin/clavulanate Piperacillin/tazobactam and ticarcillin/clavulanate Ceftolozane/tazobactam and ceftazidime/avibactam Antimicrobial resistance mechanisms Efflux Involves efflux pumps Proteins located in the bacterial cell membrane that transport molecules out of the cell Increase secretion of drug Five major superfamilies based on amino acid sequence and energy source used to export their substrates Antimicrobial resistance mechanisms Biofilms Sessile bacterial communities that are irreversibly attached to a solid surface and are embedded in an exopolysaccharide matrix Biofilms are highly resistant to antimicrobial agents. Resistance is not attributed to typically acquired genetic mechanisms but instead is determined by chemical and physical characteristics of biofilm formation. Resistance to B Lactam Drugs Gram Positive Resistance to Penicillin Production of Beta Lactamase Altered PBP target Gram Negative Resistance to Penicillin Production of Beta Lactamase Altered PBP target Decreased Uptake of drug This is important to know if we want to treat organisms effectively  Resistance mechanisms for other antimicrobials Enzymati Altered Decreased Target Enzyme Efflux c target uptake overproducti modification destructio on n Beta Lactams ✔ ✔ ✔ Glycopeptide ✔ ✔ Aminoglycoside ✔ ✔ ✔ Macrolide ✔ ✔ Quinolone ✔ ✔ Antimicrobial Susceptibility Testing (AST) Performed on bacteria and fungi isolated from clinical specimens to determine which antimicrobial agents might be effective in treating infections Methods: Generally disk diffusion or dilution Susceptibility testing are based on standards published by Clinical and Laboratory and Standards Institute (CLSI) Important note: MLTs Need to distinguish pathogens from normal flora Susceptibility tests are not routinely performed on organisms isolated from an anatomic site for which they are normal inhabitants. Example: E. coli is normal in the gastrointestinal (GI) tract and therefore would not be tested when isolated from a stool. Antimicrobial susceptibility testing (AST) Indications: When there is probable cause to suspect isolate is involved in patient infection When the susceptibility against particular antimicrobials is uncertain Not performed on bacteria known to be predictably susceptible common antimicrobial agents commonly used to treat infections Example: Group A strep is considered universally susceptible to penicillin. AST – Factors to Consider 1. Body site from which the organism was isolated Susceptibility tests are NOT routinely performed on organisms isolated from an anatomic site for which they are normal inhabitants. Ex. E. coli from GI tract 2. Presence of other organisms More than two species with greater than 105 colony-forming units (CFUs)/mL suggest contamination - especially urines 3. Quality of specimen from which the organism was grown Possible contamination Ex. In Sputum and Urines 4. Host status Patient allergies Use bactericidal or bacteriostatic agents? AST – Choice of antimicrobials Labs don’t test every antimicrobial on every isolate. Depending on the type of organism, test batteries are utilized. The Clinical and Laboratory Standards Institute (CLSI) recommends the drugs that should be tested and reported for each type of specimen. The institutional pharmacy and therapeutics committee sets the list of drugs that are used in their facility Generally test batteries are divided into two groups Gram-positive battery Staphylococci, Enterococcus, others Gram-negative battery Enterics, Pseudomonas Supplemental battery may be performed in select cases (i.e., urinary tract infections, UTIs). Contains microbial agents with enhanced activity and may be used when numerous resistant bacteria are present AST – Choice of antimicrobials Sometimes, AST is not required – predictable patterns are already known and established Example: Group A Streptococcus is predictably susceptible to penicillin so routine AST is not performed on these If patient is allergic to penicillins, AST may be warranted AST – Reporting Labs don’t report every antimicrobial which is tested Generally, reports narrow drugs first before listing secondary drugs Helps prevent confusion and over-prescription Antibiotics are categorized into groups Group A – reported first Group B - Report if the/a Isolate is resistant to primary agents Patient cannot tolerate primary agent Patient has not responded to primary agents Secondary agent would be a better clinical choice for a particular infection or is useful for treating two infections. Group C – Report for same reasons as group B These antimicrobials are more potent AST – Reporting Contraindications For pediatric patients, the following antimicrobials are NOT reported due to adverse side effects: Tetracyclines Fluoroquinolones Chloramphenicol Pregnant patients Tetracyclines Fluoroquinolones AST – Reporting Example An organism may be tested for group A, B and C drugs by the lab But only a select few get reported – generally based on groupings AST – Antimicrobial stewardship Many clinical sites in Ontario participate in Antimicrobial stewardship Antimicrobial stewardship promotes the judicious use of antimicrobials to limit the development of antimicrobial resistant organisms. Antimicrobial stewardship programs support coordinated interventions designed to improve and measure the appropriate use of antimicrobials including selection, dosing, duration of therapy and route of administration. Review: Antimicrobial Stewardship | Public Health Ontario Home | SHS+UHN Antimicrobial Stewardship Program, Toronto Canada Antimicrobial Susceptibility methods Broth Agar Methods Methods Macrodilution Disc Diffusion Microdilution Disk Gradient Agar Dilution AST Standard elements 1. Organism must be in pure culture, and in logarithmic phase 2. Organism must be prepared in standard inoculum 3. Standard Incubation: ambient air, 37C, 24 hours 4. Standard Media 5. Selection of Antimicrobial Agents 6. Quality Control AST Standard elements – Standard Inoculum Inoculum must be prepared using organisms in pure culture at logarithmic growth phase Inoculum must match the density of 0.5 McFarland Standard Equates to 1.5 X 108 colony-forming units (CFUs) per milliliter The McFarland Standard is: 0.5 mL of 0.048 M BaCl2 (1.175% w/v BaCl2.H2O) 99.5 mL of 0.36 N H2SO4 Nephelometers are used to determine the turbidity AST Standard elements – Standard media Standard media is Mueller Hinton Broth (MHB) or (MHA) Agar Agar depth: 3-4mm pH: 7.2-7.4 Variations can affect aminoglycoside, macrolide and tetracycline Cation concentrations Mg and Ca ions affects Aminoclycosides Thymidine concentration Affects SXT MHA and MHB grows aerobes and facultative anaerobes well Broth methods Broths use a dilution method Designed to determine the MIC (Minimum Inhibitory Concentration) Lowest concentration of antimicrobial agent required to inhibit the growth of a bacterial isolate The MIC value obtained is compared to known values for interpretation. Note that each organism has a different interpretation criteria Results possible Susceptible Intermediate Resistant Broth methods  Broth-Macrodilution Tests Known as tube dilution MIC tests or broth macrodilution MIC Twofold serial dilution series, each containing 1 to 2 mL of microbial agent is prepared. Recommended medium for testing Mueller-Hinton (MH) broth Standard suspension of test bacteria is added to each dilution tube. Consistent bacteria concentration: 5 × 105 CFU/mL After overnight incubation at 35° C, MIC is determined. Demonstrated by the absence of turbidity Not as useful for large panels Good for occasional antibiotic not on the panel Good for fastidious organisms with high growth requirements Broth-Macrodilution Tests Broth-Microdilution Tests - Breakpoint In Breakpoint panels, only one or a few concentrations of each antimicrobial agent are tested in a single panel. Breakpoint (cutoff) Concentration of an antimicrobial agent that coincides with a susceptible or intermediate MIC breakpoint for a particular drug Commercial systems utilizes this (Ex. BD Phoenix) Less wells required Less samples required Agar Dilution Test Not routinely performed in clinical laboratories (Exceptions: gonorrhoeae and anaerobes) Shelf life of plates is short (1 week or less). Must be stored refrigerated Labor intensive Procedure Agar plates are prepared with different concentration of antimicrobial agent, based on therapeutic range. For example, if the therapeutic range for a given antimicrobial agent is 2 to 12 μg/mL, a series of agar plates containing 1, 4, 8, 16, and 32 μg/mL is prepared A standardized suspension of bacteria is inoculated onto each agar plate Plates are incubated and results interpreted as growth Disk Diffusion Test (Kirby Bauer) Principle Drug is released from an impregnated disk and diffuses through agar, creating a zone of inhibition. Pure Culture organism prepared as a 0.5McFarland Standard Streaked to provide a lawn of growth Disks are standard 6mm wide Disks impregnated with formulary drugs Media is Mueller Hinton Agar method: Disk Diffusion Test General Steps to Set up General Steps to Interpret 1. Swab a 0.5 McFarland standard 1. Read diameter of zone of inhibition by suspension of bacteria over the entire measuring with a ruler (in mm). (Ensure surface of an agar plate (MH) plus a purity plate is pure, and acceptable) plate to assess purity. 2. Compare to established zone sizes. 2. Add paper disk containing antibiotic 3. Report as Susceptible, Intermediate, concentrations. Resistant. 3. Incubate plate overnight in O2 Agar method: Disk Diffusion Test Result Interpretation: Obtain zone size – measure the diameter of the zone of inhibition to obtain zone size The point at which the amount of drug at a specific location in the medium that is unable to inhibit the growth of the test organism Related to MIC; relationship has been used to determine breakpoints Each organism and drug will have an interpretive chart – match the zone size to the interpretation Agar method: Disk Diffusion Test Additional Considerations when interpreting: Review the purity plate to assess if results can be interpreted Sometimes you will see a haze around colonies Some organisms (ex. Proteus) will swarm – ignore this (image on top) Pinpoints within zone of inhibition These may be resistant populations – Do NOT ignore (Image on the bottom) Poorly set up plates will affect results Agar method: Disk Diffusion Test Poorly set up plates: What’s wrong with these? Kirby Bauer tests - common modifications Streptococcus species Haemophilus Neisseria Organism Various species of genus Haemophilus Not routinely done description Streptococcus, influenzae; fastidious particularly pneumoniae organism which will not grow on MH agar requires extra nutrients in O2 conditions. Media Mueller Hinton with Haemophilus Test GC Agar base which Sheep Blood Medium (HTM)- supports growth of Mueller Hinton organism must be supplemented with used hematin and NAD (X and V factor) Incubation CO2 CO2 CO2 MRSA Screen Two methods are employed to detect MRSA Some strains of MRSA are slow growing, and specialized methods are employed to identify them. Use of MRSA screen agar Media includes 2-4%NaCl and oxacillin. Media is inoculated with suspect colony and incubated at 30-35C for up to 48 h Interpretation: ANY growth indicates MRSA Use of Cefoxitin disk Use of 30ug Cefoxitin disk in a standard Kirby Bauer method Interpretation: