Bacterial Growth and Control PDF

Bacterial Growth III  Viable Counting Viable (plate) counts: measurement of living, reproducing population two main ways to perform plate counts: spread-plate method pour-plate method count colonies on plates with 30–300 col...

Bacterial Growth III  Viable Counting Viable (plate) counts: measurement of living, reproducing population two main ways to perform plate counts: spread-plate method pour-plate method count colonies on plates with 30–300 colonies To obtain the appropriate colony number, the sample to be counted should always be diluted. (Figure 5.15) Figure 5.14 Control of Bacterial Growth  Controlling Growth What is your goal? – No further growth? – Lower levels below infectious dose Remove select pathogens Meet gov regulations – sterilization Microbial Characteristics and Microbial Control https://www.cdc.gov/infectioncontrol/images/Figure-v2.png Figure 7.11  Terminology Sterilization: Removal of all microbial life Commercial sterilization: Killing of Clostridium botulinum endospores Disinfection: Removal of pathogens Antisepsis: Removal of pathogens from living tissue Degerming: Removal of microbes from a limited area Sanitization: Lower microbial counts on eating utensils Biocide/Germicide: Kills microbes Bacteriostatic: Inhibiting, not killing, microbes  Bacterial populations die at a constant logarithmic rate. Figure 7.1a  Decimal Reduction Time (DRT) or D-value Minutes to kill 90% of a population at a given temperature Table 7.2  Comparison of treatments Czank et al. 2009. Retention of the Immunological Proteins of Pasteurized Human Milk in Relation to Pasteurizer Design and Practice. Pediatric Res. 66: 374-379. DHM= donor human Control of Microbial Growth II Microbial Characteristics and Microbial Control Figure 7.11  Decimal Reduction time Loading… https://www.tu-braunschweig.de/Medien-DB/ifm/decimalreductiontime.pdf  Ionizing Radiation UV light (not ionizing) X-rays and Gamma rays Damage DNA – Breaks whole strands Proteins – Breaks peptide bonds Some bacteria more resistant than others Endospores can be very resistant  Summarize Take one minute and summarize radiation as a sterilizing agent What is the general mechanism? What are the limitations of each method  Filters Second most common method of sterilizing liquids Depends on pore size or layers of fibers Membrane filters with known pore sizes are commonly used  Chemical Control Bacteriostatic – Do not kill but inhibit growth – Immune system does rest! Bacteriocidal – Kills bacteria, but may not kill other microorganisms Bacteriolytic – Kill bacteria by lysing cell Viability Staining using Confocal laser-scanning microscopy Loading…  Effectiveness: Minimum Inhibitory Concentration  Chemical Methods of Control Evaluating a disinfectant – Disk-diffusion method Figure 7.6 Automated Reader  Disk Diffusion Assay What are the advantages to this assay? What might be the disadvantages?  Summarize Bacteriostatic Bacteriolytic Bacteriocidal What is minimum inhibitory concentration? What is disk diffusion? How is a Zone of inhibition related to MIC? Antimicrobial Drugs  Combinations of antimicrobial drugs Synergistic: When action of one drug enhances another Antagonistic: When action of one drug Loading… interferes with another Additive: When effect of combination is neither synergistic or antagonistic  Mechanisms of Action of Antibacterial Drugs Mechanism of action include: – Inhibition of cell wall synthesis – Inhibition of protein synthesis – Inhibition of nucleic acid synthesis – Inhibition of folate biosynthesis pathways – Interference with cell membrane integrity  In summary List the 4 major classes of cell wall inhibitors. What do penicillins and Cephalosporins have in common? How do they generally work?  Why do sulfonamides work? Discuss how the metabolic pathway inhibitors sulfonamides and trimethoprim are selectively toxic.  Antibiotic Resistance end of a Golden Age? The potential for largest medical crisis in your near future!!!!!!  Introduce antibiotic resistance – One of the largest medical crises you will potentially face! – Just one Example: – About 440 000 new cases of multidrug-resistant tuberculosis (MDR-TB) annually – 150 000 deaths (34%) – Extensively drug-resistant tuberculosis (XDR-TB) has been reported in 64 countries to date.  MDRO the new bad guys Multi-Drug Resistant Organisms MRSA, VRSA, VRE Gram negative bacteria with Extended Loading… spectrum beta-lactamases – E.g. E. coli and Klebsiella pneumoniae MRSA (data from CDC) – 1968 first isolated – 1990s 20-25% – 1999 >50% – 2003 69%  – Current 1 in 3 carry S. aureus, 1 in 50 carry MRSA Determining Susceptibility of Bacterial to Antimicrobial Drug Susceptibility of organism to specific antimicrobials is unpredictable Often drug after drug tried until favorable response was observed – If serious infection, several drugs may be prescribed at one time with hope that one was effective Better approach – determine susceptibility – Prescribe drug that acts against offending organism Best to choose one that affects as few others as possible  Determining Susceptibility of Bacterial to Antimicrobial Drug Determining MIC – MIC = Minimum Inhibitory Concentration Quantitative test to determine lowest concentration of Loading… specific antimicrobial drug needed to prevent growth of specific organism – Determined by examining strain’s ability to grow in broth containing different concentrations of test drug  Determining Susceptibility of Bacterial to Antimicrobial Drug MIC is determined by producing serial dilutions with decreasing concentrations of test drug Known concentrations of organism is added to each test tube Tubes are incubated and examined for growth – Growth determined by turbidity of growth medium Lowest concentration to prevent growth is MIC  Determining Susceptibility of Bacterial to Antimicrobial Drug Conventional disc diffusion method – Kirby-Bauer disc diffusion routinely used to qualitatively determine susceptibility – Standard concentration of strain uniformly spread of – Clear zone of inhibition standard media around disc reflects – Discs impregnated with susceptibility specific concentration of Based on size of zone antibiotic placed on plate organism can be described and incubated as susceptible or resistant  What do you remember? What is susceptible? What is resistance? What is Minimum Inhibitory Concentration? Disc diffusion assay?  Resistance to Antimicrobial Drugs Mechanisms of resistance – Drug inactivating enzymes Some organisms produce enzymes that chemically modify drug Loading… – Penicillinase breaks β-lactam ring of penicillin antibiotics – Alteration of target molecule Minor mutations that change an antibiotic target can prevent binding – Changes in ribosomal RNA prevent macrolids from binding to ribosomal subunits  Determining Susceptibility of Bacterial to Antimicrobial Drug Mechanisms of resistance – Decreased uptake of the drug Alterations in porin proteins decrease permeability of cells – Prevents certain drugs from entering – Increased elimination of the drug Some organisms produce efflux pumps – Increases overall capacity of organism to eliminate drug » Enables organism to resist higher concentrations of drug » Tetracycline resistance Innate resistance = no target of drug  Take note What are the 5 major mechanisms of drug resistance? How do they infer resistance?  Determining Susceptibility of Bacterial to Antimicrobial Drug Acquisition of resistance – Can be due to spontaneous mutation Alteration of existing genes Spontaneous mutation called vertical evolution – Or acquisition of new genes Resistance acquired by transfer of new genes called horizontal transfer  Determining Susceptibility of Bacterial to Antimicrobial Drug Spontaneous mutation – Occurs at relatively low rate – Such mutations have profound effect of resistance of bacterial population – Example of spontaneous mutation Resistance to streptomycin is result a change in single base pair encoding protein to which antibiotic binds – When antimicrobial has several different targets it is more difficult for organism to achieve resistance through spontaneous mutation  Determining Susceptibility of Bacterial to Antimicrobial Drug Acquisition of new genes through gene transfer – Most common mechanism of transfer is through conjugation Transfer of R plasmid Plasmid often carries several different resistance genes – Each gene mediating resistance to a specific antibiotic » Organism acquires resistance to several different drugs simultaneously  Determining Susceptibility of Bacterial to Antimicrobial Drug Examples of emerging antimicrobial resistance – Enterococci Part of normal intestinal flora Common cause of nosocomial infections Intrinsically resistant to many common antimicrobials Some strains resistant to vancomycin – Termed VRE » Vancomycin resistant enterococcus Many strains achieve resistance via transfer of plasmid  Determining Susceptibility of Bacterial to Antimicrobial Drug Staphylococcus aureus – Common cause of nosocomial infections – Becoming increasingly resistant In past 50 years most strains acquired resistance to penicillin – Due to acquisition of penicillinase genes Until recently most infections could be treated with methicillin (penicillinase resistant penicillin) – Many strains have become resistant » MRSA methicillin resistant Staphylococcus aureus MRSA many of these strains still susceptible to vancomycin – Some hospitals identified VISA » VISA vancomycin intermediate Staphylococcus aureus Mechanisms of Horizontal Transfer Conjugation – transfer through sex pilus – Requires plasmid for sex pilus formation – Can transfer plasmids or portions of the chromosomes (recombination) Transformation – picking foreign genes from environment – Plasmids lost by other bacteria Transduction – movement of genes by viruses – Accidental movement – Picks up chromosomal DNA and moves to new  host Determining Susceptibility of Bacterial to Antimicrobial Drug Streptococcus pneumoniae – Has remained sensitive to penicillin Some strains have now gained resistance – Resistance due to modification in genes coding for penicillin- binding proteins » Changes due to acquisition of chromosomal DNA from other strains of Streptococcus » Generally via DNA mediated transformation  Determining Susceptibility of Bacterial to Antimicrobial Drug Slowing emergence and spread of resistance – Responsibilities of physicians and healthcare workers Increase efforts to prescribe antibiotics for specific organisms Educate patients on proper use of antibiotics – Responsibilities of patients Follow instructions carefully Complete prescribed course of treatment – Misuse leads to resistance  Determining Susceptibility of Bacterial to Antimicrobial Drug Slowing emergence and spread of resistance – Importance of an educated public Greater effort made to educate public about appropriateness and limitations of antibiotics – Antibiotics have no effect on viral infections – Misuse selects antibiotic resistance in normal flora – Global impacts of the use of antimicrobial drugs Organisms develop resistance in one country can be transported globally Many antimicrobials are available as non-prescription basis Use of antimicrobials drugs added to animal feed – Produce larger more economically productive animals – Also selects for antimicrobial resistant organisms  CDC recommendations for Reduction of Antimicrobial resistance in Healthcare Infection prevention Accurate and prompt diagnosis and treatment Prudent use of antimicrobials Prevention of transmission (www.cdc.gov/drugresistance/healthcare/default.htm)  Health Care Recommendations Prevent Infection – Vaccinate – Get the catheters out Diagnose and Treat Infection Effectively – Target the pathogen – Access infectious disease experts Use Antimicrobials Wisely – Practice antimicrobial control  – Use local data—know your patient How can you do it? Discuss with your Neighbor – What is a major cause of antibiotic resistance – What is one way you can help prevent the creation of potential antibiotic resistant strains of bacteria?  Metabolism I You are what you eat or energy conservation R us Microbial Metabolism Metabolism: The sum of the chemical reactions in an organism Catabolism: The energy-releasing processes Anabolism: The energy-using processes Microbial Metabolism Catabolism provides the building blocks and energy for anabolism. Loading… Figure 5.1  A metabolic pathway is a sequence of enzymatically catalyzed chemical reactions in a cell. Metabolic pathways are determined by enzymes. Enzymes are a type of protein encoded by genes. Loading… Two major Pathways for Energy Conservation Respiration Pathways = Glycolysis and electron transport Complete oxidation to CO2 High ATP production Exogenous electron acceptor Fermentation Pathways = Glycolysis (Embden-Meyerhof pathway) and Fermentation Partial oxidation Low ATP production Endogenous electron acceptor Energy Conservation / conversion Energy containing molecules oxidized to release energy Fire – oxidize carbohydrates uses oxygen as electron acceptor CO2 and H2O + lots of heat (random energy) Metabolic Pathways – oxidize carbohydrates CO2 and H2O Some heat (random energy) Step wise utilization of energy Energy conserved as Electron carriers and ATP Aerobic pathways use oxygen as electron acceptor Chemoorganotropic ATP production Respiration Pathways – use Exogenous electron acceptors Electrons from organic molecules (often sugars) Electrons to electron transport to make proton motive force (chemical gradient) Proton motive force used to make ATP (oxidative phosphorylation) – high ATP Final Electron acceptor is oxygen Anerobic – Electrons from organic molecules (often sugars) Electrons to electron transport to make proton motive force (chemical gradient) Proton motive force used to make ATP (oxidative phosphorylation) – high ATP Final Electron acceptor is something besides oxygen (not oxygen) Fermentation Pathways – use Endogenous electron acceptors Electrons from organic molecules Make ATP through Substrate Level Phosphorylation Reduce an organic molecule from the Fermentation pathway Chemoorganotrophic ATP production Fermentation products: Ethanol, lactic acid Endogenous e- Acceptor: like pyruvate Catabolic Reactions Synthesis of ATP driven by oxidation and reduction reactions Energy Conserved: Short-term as ATP or Electron carriers Long-term as chemical potential. Examples? Final Amount of energy from substrate depends on: State of substrate Final electron acceptor Proton Motive Force Central Energy Conservation Mechanism Chemiosmosis Often linked to electron transport system Loading… Drives Oxidative Phosphorylation Drives membrane transport Drives locomotion Proton Motive Force What is it? Creation of chemical potential (or) Creation of proton or pH gradient Energy from Oxidation and reduction Electron transport proteins Embedded in Cell membrane What for? Locomotion Oxidative phosphorylation Active transport Where does the energy originate? Oxidation of reduced organic molecules Often carbohydrates or lipids Usually in form electron carriers/transporters Chemoorganotrophic ATP production Fermentation products: Ethanol, lactic acid Endogenous e- Acceptor: like pyruvate Metabolic Choice: Is there an exogenous electron acceptor? Exogenous e- acceptor  Exogenous Electron Acceptor TCA Cycle Figure 5.13 - Overview Figure 5.14 (2 of 2) Figure 5.16 (2 of 2) Figure 5.15 (2 of 2) Table 5.3  Next Time Fermentation and metabolic diversity Homework: Chapter 5 Outline Glycolysis – substrate and product Outline Krebs – substrate and product Outline electron transport Anerobic and Aerobic Substrate and products Created by Turbolearn AI Page 1  Created by Turbolearn AI adenosinetriphosphate Page 2  Created by Turbolearn AI upto36AT P e. g. sulfate, nitrate usually2 − 4AT P 2AT P usesendogenouselectronacceptor Page 3 Created by Turbolearn AI Page 4 Created by Turbolearn AI Page 5 Created by Turbolearn AI Page 6 Created by Turbolearn AI Page 7 Created by Turbolearn AI Page 8 Created by Turbolearn AI Page 9 Created by Turbolearn AI Page 10 Created by Turbolearn AI Page 11 Created by Turbolearn AI Page 12 Created by Turbolearn AI Page 13 21. What is artificial selection, and give an example? Answer: Artificial selection is when humans choose which organisms survive and reproduce, such as selecting crops resistant to pests. 22. What is the purpose of using Thayer-Martin agar? Answer: It is used to isolate Neisseria gonorrhoeae by inhibiting the growth of contaminants. 23. Define natural selection. Answer: Natural selection is the process where the environment determines which organisms survive and reproduce. 24. What is the primary difference between selective and differential media? Answer: Selective media inhibit unwanted organisms, while differential media distinguish organisms based on biochemical characteristics. 25. Name an example of a medium that is both selective and differential. Answer: MacConkey agar, which is selective for Gram-negative bacteria and differentiates lactose fermenters. 26. What is the role of hemolysin in microbial growth on blood agar? Answer: Hemolysin breaks down red blood cells, which can be visualized as clear zones around colonies. 27. What are viable plate counts used for in microbiology? Answer: To measure living, reproducing populations of microorganisms. 28. How do you identify microbial growth using metabolites? Answer: By detecting specific byproducts, such as acids or gases, produced during microbial metabolism. 29. What are some factors influencing microbial population changes in artificial selection? Answer: Environmental stressors like antibiotics, herbicides, and selective breeding for specific traits. 30. What is the purpose of using counting chambers in microbiology? Answer: To calculate cell numbers in a defined volume for precise microbial enumeration. 51. What is the purpose of using Mannitol Salt Agar? Answer: It selects for osmotolerant organisms and differentiates those that ferment mannitol. 52. What are the two main types of selection in biological systems? Answer: Artificial selection (human-driven) and natural selection (environment-driven). 53. Give an example of artificial selection using antibiotics. Answer: Antibiotics select for resistant strains of bacteria by killing susceptible ones. 54. What is the function of a pH indicator in MacConkey agar? Answer: It differentiates bacteria based on their ability to ferment lactose, changing color in response to acid production. 55. Explain the importance of selective media in microbiology. Answer: Selective media suppress unwanted microbes and allow the growth of specific organisms, aiding in their isolation and study. 56. What is a counting chamber, and how is it used? Answer: A counting chamber is a device with a defined volume and grid used to count microbial cells under a microscope. 57. How do bile salts function in MacConkey agar? Answer: They inhibit Gram-positive bacteria, making the medium selective for Gram-negative bacteria. 58. What is the role of hemolysins in pathogenic bacteria? Answer: Hemolysins allow bacteria to break down red blood cells, often contributing to their virulence. 59. Why is it important to dilute a sample before viable plate counting? Answer: Dilution ensures that colony counts fall within a countable range of 30–300 colonies per plate. 60. What is the significance of the decimal reduction time (D-value) in sterilization? Answer: It helps determine the time required to achieve a specific level of microbial reduction, ensuring the effectiveness of sterilization methods. 1. Application: How could selective media help reduce contamination in healthcare settings? Answer: By isolating and identifying pathogens quickly, enabling targeted infection control measures. 2. Analysis: Why might artificial selection in agriculture lead to unintended consequences, like reduced crop diversity? Answer: Artificial selection focuses on specific traits, potentially neglecting genetic variation needed for resilience. 3. Evaluation: Evaluate the impact of antibiotic selection pressure on hospital-acquired infections. Answer: It promotes resistant strains, increasing treatment difficulty and healthcare costs. 4. Application: How could MacConkey agar be used to monitor sanitation in food processing plants? Answer: It can identify Gram-negative bacteria, which often indicate contamination or poor hygiene. 5. Synthesis: Propose a plan to study microbial resistance evolution in urban environments. Answer: Collect samples 21. Application: How could the principles of artificial selection be applied to develop crops resistant to specific fungal pathogens? Answer: Select and crossbreed plants showing natural resistance to the pathogen, then test offspring for durability and yield. 22. Analysis: Why might selective media fail to identify pathogens in complex environmental samples like soil? Answer: Soil contains diverse microbes and substances that could interfere with the media’s selectivity and inhibit target organism growth. 23. Evaluation: Assess the effectiveness of pasteurization in preventing foodborne illnesses compared to refrigeration. Answer: Pasteurization kills pathogens, reducing immediate risks, but refrigeration slows microbial growth and is essential for long-term preservation. 24. Application: How can you use differential media to identify drug-resistant bacteria in hospital wastewater? Answer: Use media that differentiate resistant strains (e.g., with antibiotics incorporated) and compare growth patterns to non-resistant controls. 25. Synthesis: Design a method to reduce the spread of antibiotic-resistant bacteria in livestock farming. Answer: Implement selective breeding, reduce antibiotic use, introduce probiotics, and regularly monitor microbial populations with selective media. 26. Analysis: Why might biofilm formation in water distribution systems pose a challenge to public health? Answer: Biofilms protect pathogens from disinfectants and can release harmful microbes into the water supply. 27. Evaluation: Compare the advantages and disadvantages of using selective vs. non-selective media in environmental monitoring. Answer: Selective media isolate specific organisms, but non-selective media provide a broader picture of microbial diversity. 28. Application: How could differential media be used to identify spoilage organisms in canned food? Answer: Use media that detect metabolic byproducts like acid or gas production, indicative of spoilage activity. 29. Synthesis: Develop a protocol for testing the effectiveness of different antiseptics on wound bacteria using selective media. Answer: Inoculate selective media with wound samples, treat with antiseptics, and compare colony growth and type before and after treatment. 30. Evaluation: Critique the use of MacConkey agar for diagnosing urinary tract infections (UTIs) in remote clinics. Answer: While effective for isolating Gram-negative pathogens, it may require complementary tests for comprehensive diagnosis.