Microbiology: Inhibiting the Growth of Microorganisms in Vitro Class 4

1. Microbiology:Inhibiting the Growth of Microorganisms in VitroClass 4

2. Definition of Terms • Sterilization is the complete destruction of all microorganisms, including cells, spores and viruses. • Accomplished by dry heat, autoclaving (steam under pressure), gas, various chemicals and certain types of radiation. • Sanitization is the reduction on microbial populations to those considered safe by public health standards • Disinfection is the destruction or removal of pathogens from nonliving objects by physical or chemical methods; example = pasteurization. • Disinfectants are chemical substances that eliminate pathogens on inanimate objects. • Antiseptics are solutions used to disinfect skin and other living tissues.

3. Definition of Terms • The suffix –cide or –cidal refers to “killing.” • Germicidalagents, biocidal agents and microbicidal agents are chemicals that kill microbes. • Bactericidal agents are chemicals that specifically kill bacteria, but not necessarily endospores. • Sporicidal agents kill bacterial endospores. • Fungicidal agents kill fungi, including fungal spores. • Algicidal agents kill algae. • Viricidal agents destroy viruses.

4. Definition of Terms (cont.) • A microbistatic agent is a drug or chemical that inhibits growth and reproduction of microorganisms. • A bacteriostatic agent is one that specifically inhibits the metabolism and reproduction of bacteria. • Lyophilization is a process that combines dehydration (drying) and freezing. This process is widely used in industry to preserve foods, antibiotics, microorganisms and other biologic materials. • Sepsisrefers to the presence of pathogens in blood or tissues, whereas asepsis means the absence of pathogens. • Asepsis refers to the absence of pathogens • Antisepsis is the prevention of infection.

5. Physical Methods to Inhibit Microbial Growth • Heat • 2 factors – temperature and time - determine the effectiveness of heat for sterilization. • The thermal death point (TDP) of any species is the lowest temperature that will kill all of the organisms in a standardized pure culture within a specified time. • Types of Heat • Dry heat – flame or electrical incinerator • Moist heat – boiling or use of an autoclave

6. Physical Methods to Inhibit Microbial Growth (cont.) • The autoclave • A large metal pressure cooker that uses steam under pressure to completely destroy all microbial life. • Increased pressure raises the temperature above the temperature of boiling water (above 100oC) and forces steam into material being sterilized. • Autoclaving at a pressure of 15 psi at 121.5oC for 20 minutes kills vegetative microorganisms, bacterial endospores and viruses. • Can use pressure-sensitive tape or spore strips as a quality control measure to ensure proper autoclaving.

7. Physical Methods to Inhibit Microbial Growth (cont.) • Cold; most microorganisms are not killed, but their metabolic activities are slowed. • Desiccation; many dried microorganisms remain viable, but they cannot reproduce. • Radiation; an ultra-violet (UV) lamp is useful for reducing the number of microorganisms in the air. • Ultrasonic waves; used in hospitals and medical and dental clinics to clean and sterilize equipment. • Filters; used to separate cells/microorganisms from liquids or gases. • Gaseous atmosphere; can be altered to inhibit growth.

8. Using Chemical Agents to Inhibit Microbial Growth • Chemical disinfection refers to the use of chemical agents to inhibit the growth of pathogens, either temporarily or permanently. • Disinfectants are affected by: • Prior cleaning of the object or surface • The organic load (e.g., feces, blood, pus) • The bioburden; type and number of microbes • Concentration of disinfectant • Contact time • Physical nature of the object being disinfected • Temperature and pH

10. Should have a broad antimicrobial spectrum Fast acting Not affected by the presence of organic matter Nontoxic to human tissues and noncorrosive Should leave a residual antimicrobial film on surface Soluble in water and easy to apply Inexpensive and easy to prepare Stable as both a concentrate and a working solution Odorless Using Chemical Agents to Inhibit Microbial Growth (cont.) Characteristics of an ideal chemical antimicrobial agent:

11. Using Chemical Agents to Inhibit Microbial Growth (cont.) • Antiseptics • May safely be used on human tissues. • Reduce the number of organisms on the surface of the skin; do not penetrate pores and hair follicles. • Antiseptic soaps and scrubbing are used by healthcare personnel to remove organisms lodged in pores or folds of the skin.

12. Inhibiting the Growth of Pathogens in Our Kitchens • Many foods brought into our kitchens are contaminated with pathogens; examples = E. coli 0157:H7, Salmonella and Campylobacter spp. on poultry and ground beef. • Problems arise when handling raw foods before cooking. • Remain aware of pathogens when preparing foods. • Wash hands frequently. • Thoroughly clean plates that have had poultry or meat on them with hot soapy water or place in a dishwasher. • Use an antibacterial kitchen spray.

13. Controversies Relating to the Use of Antimicrobial Agents in Animal Feed and Household Products • 40% of the antibiotics manufactured in the U.S. are used in animal feed; microorganisms resistant to these antibiotics survive! • Drug resistant organisms are transmitted in animal feces and in food products. • Efforts are underway to eliminate or reduce the practice of adding antibiotics to animal feed. • Use of antimicrobial agents is widespread in toys, cutting boards, in hand soaps and many other household products; resistant microorganisms survive! • Controversy: Should children be exposed to all sorts of microorganisms for their immune systems to develop properly?

14. Antimicrobial agents • Chemotherapy is the use of any chemical (drug) to treat any disease or condition. • A chemotherapeutic agent is any drug used to treat any condition or disease. • An antimicrobial agentis any chemical (drug) used to treat an infectious disease, either by inhibiting or killing pathogens. • An antibiotic is a substance produced by a microorganism that kills or inhibits growth of other microorganisms (usually bacteria). • Antibiotics that have been chemically modified to kill a wider variety of pathogens or reduce side effects are called semisynthetic antibiotics; examples include semisynthetic penicillins such as ampicillin and carbenicillin.

15. Ideal Qualities of an Antimicrobial Agent • The ideal antimicrobial agent should: • Kill or inhibit the growth of pathogens. • Cause no damage to the host. • Cause no allergic reaction in the host. • Be stable when stored in solid or liquid form. • Remain in specific tissues in the body long enough to be effective. • Kill the pathogens before they mutate and become resistant to it.

16. How Antimicrobial Agents Work • The 5 most common mechanisms of action of antimicrobial agents are: • Inhibition of cell wall synthesis. • Damage to cell membranes. • Inhibition of nucleic acid synthesis (either DNA or RNA synthesis). • Inhibition of protein synthesis. • Inhibition of enzyme activity.

17. Antibacterial Agents • Sulfonamide drugs inhibit production of folic acid (a vitamin) in those bacteria that require p-aminobenzoic acid to synthesize folic acid; without folic acid bacteria cannot produce certain essential proteins and die. • Sulfa drugs are competitive inhibitors; they are bacteriostatic. • In most Gram-positive bacteria, penicillin interferes with the synthesis and cross-linking of peptidoglycan, a component of cell walls. By inhibiting cell wall synthesis, penicillin destroys the bacteria.

18. Antibacterial Agents (cont.) • Colistin and nalidixic acid destroy only Gram-negative bacteria; they are referred to as narrow-spectrum antibiotics. • Antibiotics that are destructive to both Gram-positive and Gram-negative bacteria are called broad-spectrum antibiotics (examples: ampicillin, chloramphenicol and tetracycline). • Multidrug therapy • Sometimes one drug is not sufficient; 2 or more drugs may be used simultaneously, as in the treatment of tuberculosis.

19. Antibacterial Agents (cont) • Synergism Versus Antagonism • Synergismis when 2 antimicrobial agents are used together to produce a a degree of pathogen killing that is greater than that achieved by either drug alone. Synergism is good! • Antagonism is when 2 drugs actually work against each other. The extent of pathogen killing is less than that achieved by either drug alone. Antagonism is bad!

20. Antifungal Agents • Most antifungal agents work in one of 3 ways: • By binding with cell membrane sterols (e.g., nystatin and amphotericin B). • By interfering with sterol synthesis (e.g., clotrimazole and miconazole). • By blocking mitosis or nucleic acid synthesis (e.g., griseofulvin and 5-flucytosine).

21. Antiprotozoal Agents • Antiprotozoal agents are usually toxic to the host. • Antiprotozoal agents work by: • Interfering with DNA and RNA synthesis (e.g., chloroquine, pentamidine and quinacrine). • Interfering with protozoal metabolism (e.g., metronidazole).

22. Antiviral Agents • Antiviral agents are the newest weapons in antimicrobial methodology. • Difficult to develop these agents because viruses are produced within host cells. • Some drugs have been developed that are effective in certain viral infections, but not others; they work by inhibiting viral replication within cells. • Antiviral agent “cocktails” (drugs that are administered simultaneously) are being used to treat HIV infection.

23. Drug Resistance“Superbugs” • Superbugs are microorganisms (mainly bacteria) that have become resistant to one or more antimicrobial agent. Infections caused by superbugs are difficult to treat! • Superbugs include: • methicillin-resistant Staphylococcus aureus (MRSA); vancomycin-resistant Enterococcus spp. (VRE); multidrug-resistant Mycobacterium tuberculosis (MRTB); multidrug-resistant strains of Pseudomonas spp., Stenotrophomonas spp., Salmonella spp., Shigella spp. and N. gonorrhoeae; and β–lactamase-producing strains of Streptococcus pneumoniae and Haemophilus influenzae.

24. Drug ResistanceHow Bacteria Become Resistant to Drugs • Before a drug enters a bacterial cell it must first bind to proteins on the surface of the cell; these proteins are called drug-binding sites. • Some are naturally resistant because they lack the specific target site for the drug. • In intrinsic resistancethe drug is unable to cross the organism’s cell wall or cell membrane and thus, cannot reach its site of action. • If bacteria that were once susceptible to a particular drug become resistant, this is called acquired resistance.

25. Drug ResistanceHow Bacteria Become Resistant to Drugs (cont.) • To enter a bacterial cell, a drug must be able to pass through the cell wall and cell membrane; chromosomal mutations may alter the structure of the cell membrane causing the organism to become resistant. • Bacteria can develop the ability to produce an enzyme that destroys or inactivates a drug. • Many bacteria have become resistant to penicillin because they have acquired the gene for penicillinase production during conjugation.

26. Drug ResistanceHow Bacteria Become Resistant to Drugs (cont.) • Bacteria can also become resistant to drugs by developing the ability to produce multidrug-resistance (MDR) pumps which enables the cell to pump out drugs before they can damage or kill the cell. • Summary: Bacteria can acquire resistance to antimicrobial agents by chromosomal mutation or the acquisition of new genes by transduction, transformation and, most commonly, by conjugation.

27. Drug Resistanceβ-Lactamases • Every penicillin and cephalosporin molecule contains a double-ringed structure known as the β-lactam ring. • Some bacteria produce enzymes, β-lactamases, that destroy this ring; when the β–lactam ring is destroyed, the drug no longer works. • 2 types of β-lactamases - penicillinases and cephalosporinases; some bacteria produce both types. • Drug companies have developed special drugs that combine a β–lactam antibiotic with a β-lactamase inhibitor.

28. Education of healthcare professionals and patients. Patients should stop demanding antibiotics every time they or their child is sick. Physicians should not be pressured by patients and should prescribe drugs only when warranted. Clinicians should prescribe a narrow-spectrum drug if lab results indicate that it kills the pathogen. Patients should destroy any excess or out-dated medications. Antibiotics should not be used in a prophylactic manner. Healthcare professionals should practice good infection control. Patients should take drugs in manner prescribed. What Clinicians and Patients Can Do To Help in the War Against Drug Resistance

29. Empiric Therapy • Empiric therapy is when drug therapy is initiated before laboratory results are available (i.e., before the pathogen is identified and/or before susceptibility test results are available). • Empiric therapy is sometimes necessary to save a patient’s life. • Clinicians make an “educated guess” based on past experience with infectious diseases and the most effective drugs. • Clinicians must take a number of factors into consideration before prescribing antimicrobial agents.

30. If pathogen identity is known, use lab’s chart of antimicrobial susceptibility test data from past year. Is the patient allergic to any antimicrobial agents? What is the age of the patient? Is the patient pregnant? Inpatient or outpatient? In the hospital formulary? Site of the infection? What other medication is the patient taking? What other medical problems does the patient have? Is the patient leukopenic or immunocompromised? What is the cost of the drug(s)? Empiric Therapy (cont.)Factors to be Considered

31. Undesirable Effects of Antimicrobial Agents • Reasons why antimicrobial agents should not be used indiscriminately: • Organisms susceptible to the agent will die, but resistant ones will survive; this is known as selecting for resistant organisms. • The patient may become allergic to the agent. • Many agents are toxic to humans and some are very toxic. • With prolonged use, a broad-spectrum antibiotic may destroy the normal flora resulting in an overgrowth of bacteria known as a superinfection due to decreased microbial antagonism.

32. Epidemiology • Introduction • Epidemiology can be defined as the study of disease. • Epidemiologists study the factors that determine the frequency, distribution and determinants of diseases in human populations. • Epidemiologists also develop ways to prevent, control or eradicate diseases in populations.

33. Epidemiologic Terminology • An infectious disease is a disease caused by the entrance into the body of organisms (as bacteria, protozoans, fungi, or viruses) which grow and multiply there • A communicable diseaseis an infectious disease that can be transmitted from one person to another • A contagious diseaseis a communicable disease that is easily transmitted from person-to-person. • A zoonotic disease is one that humans acquire from animal sources. • The incidence of a particular disease is the number of new cases of that disease in a defined population during a specific time period.

34. Epidemiologic Terminology (cont.) • An Acute illness is sudden onset and lasts for a short time • A Chronic illness comes on slowly and lasts a long time • A Terminal illness is a disease with no potential for cure • An Idiopathic disease is one with no known cause • A Latrogenic disease is cause by adverse effects of treatment.

35. Epidemiologic Terminology (cont.) • Prevalence • Period prevalenceis the number of cases of the disease existing in a given population during a specific time period (e.g., during the year 2006). • Point Prevalenceis the number of cases of the disease existing in a given population at a particular moment in time. • Morbidity rate is the number of new cases of a particular disease that occurred during a specified time period per a specifically defined population (usually per 1,000, 10,000 or 100,000 population). • Mortality rateis the ratio of the number of people who died of a particular disease during a specified time period per a specified population

36. Epidemiologic Terminology (cont.) • A sporadic diseaseis one that occurs only occasionally within the population of a particular geographic area; example, tetanus. • An endemic diseaseis one that is always present within the population of a particular geographic area; example, gonorrhea. • An epidemic diseaseis defined as a greater than usual number of cases of a disease in a particular region, usually within a short period of time; example, the Legionnaire’s disease epidemic of 1976.

37. Epidemiologic Terminology (cont.) • A pandemic is a disease that is occurring in epidemic proportions in many countries or a continent simultaneously. Examples include: • Influenza • Example, the Spanish flu pandemic of 1918; more than 20 million people were killed worldwide (500,000 in the U.S.) • HIV/AIDS • Tuberculosis • Malaria

38. Interactions Among Pathogens, Hosts, and the Environment • Whether an infectious disease occurs depends on: • Factors pertaining to the pathogen (e.g., virulence of pathogen, mode of entry, number of organisms). • Factors pertaining to the host; these include health status, nutritional status, hygiene, age, travel, lifestyle etc. • Factors pertaining to the environment such as physical factors (e.g., climate, season, geographic location), availability of appropriate reservoirs, sanitary and housing conditions, and availability of potable water.

39. Chain of Infection There are 6 components in the infectious disease process: ● a pathogen ● a source of the pathogen (a reservoir) ● a portal of exit ● a mode of transmission ● a portal of entry ● a susceptible host

40. Reservoirs of Infection • The sources of microorganisms that cause infectious diseases are many and varied; they are known as reservoirs of infection or simply reservoirs. • Living reservoirs – humans, pets, farm animals, insects, arachnids. • Human carriers: • Passive carriers- carry the microorganism but do not have the disease • Incubatory carriers- Capable of transmitting the disease during the incubatory stage of a disease • Convalescent carriers- Capable of transmitting the disease during the recovery period (incubatory) stage of a disease • Active carriers- are healed from the active disease but still capable of transmitting the disease

41. Reservoirs of Infection (cont.) • Animals • Infectious diseases that humans acquire from animal sources are called zoonotic diseasesor zoonoses. • Zoonotic diseases may be acquired by direct contact with an animal, inhalation or ingestion of the pathogen, or injection of the pathogen by an arthropod. • Examples: • Rabies • Lyme disease

42. Reservoirs of Infection (cont.) • Arthropods • Many different types of arthropods serve as reservoirs of infection, including insects (e.g., fleas, mosquitoes, lice) and arachnids (e.g., mites and ticks) • When arthropods are involved in the transmission of infectious diseases they are referred to as vectors. • Examples of arthropod-borne diseases: • Lyme disease • Malaria

43. Reservoirs of Infection (cont.) • Nonliving Reservoirs • Air, soil, dust, contaminated water and foods, insects, and infected humans. • Fomites - inanimate objects capable of transmitting pathogens (e.g., bedding, towels, eating and drinking utensils, hospital equipment, etc.)

44. Modes of Disease Transmission

45. Modes of Transmission • Direct skin-to-skin contact • Direct mucous membrane-to-mucous membrane contact by kissing or sexual intercourse • Indirectly by airborne droplets of respiratory secretions, usually by sneezing or coughing • Indirectly by contamination of food and water by fecal matter • Indirectly by arthropod vectors • Indirectly by contaminated fomites • Indirectly by transfusion of contaminated blood or blood products or by parenteral injection using nonsterile syringes or needles

46. Public Health Agencies • World Health Organization (WHO) • A specialized agency of the United Nations founded in 1948; www.who.org • Mission: to promote technical cooperation for health among nations, carry out programs to control and eradicate diseases and improve the quality of human life. • Investigates outbreaks of Ebola virus, etc. • Eradicated smallpox • Attempting to eradicate polio and dracunculiasis

47. Public Health Agencies (cont.) • Centers for Disease Control and Prevention (CDC) • A federal agency administered by the U.S. Department of Health and Human Services, Atlanta, GA. Established in 1946; www.cdc.gov • Mission: “to promote health and quality of life by preventing and controlling diseases, injury, and disability …” • Certain infectious diseases, known as nationally notifiable diseases must be reported to the CDC.(TB, West Nile virus, Lyme disease, Pertussis, Syphilis) • Publishes Morbidity and Mortality Weekly Report (MMWR).

48. Public Health Agencies (cont.) • Measures for prevention and control of epidemics: • Increase host resistance through the development and administration of vaccines that induce active immunity and maintain it in susceptible persons. • Ensure that persons exposed to a pathogen are protected against the disease. • Segregate, isolate and treat those who have contracted a contagious infection to prevent the spread of the pathogen to others. • Identify and control potential reservoirs and vectors of infectious diseases.

49. Bioterrorism and BiologicalWarfare Agents • Microbes purposely used to harm others in wartime are called biological warfare (bw) agents. • Pathogens used to create fear, chaos, illness and death are called bioterrorism agents. Examples: • Bacillus anthracis • Clostridium botulinum • Smallpox virus (Variola major) • Yersinia pestis

50. Water Supplies and Sewage Disposal • Water is the most essential resource for the survival of humanity! • Sources of water contamination: • rainwater and groundwater (from wells) can become contaminated by soil microbes and raw fecal material.