Bio 420 - Lecture 08 - Chapters 17 & 20 PDF

BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Part III - Microbes & Humans Lecture 8 Chapter 17. Applications o...

BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Part III - Microbes & Humans Lecture 8 Chapter 17. Applications of Immune Responses Chapter 20. Antimicrobial Medications Anderson et al., Nester’s Microbiology: A Human Perspective. McGraw-Hill Publishing, Edition 10e, 2020 Animated Denotes animated slide that can be viewed in “Slide Show” mode Denotes explanations given in the slide notes that can be viewed via “View  Note page” See slide notes or printed via “Print  Note page” VIDEO Denotes the links to video clips given in slide notes 7/26/2024 Animated VIDEO BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 WWW See slide notes 1 To print the slide notes, choose “Notes Pages” from PRINT menu. To see the slide notes on computer screen, choose “Notes Page” from VIEW menu To run presentation, select “Slide Show” from right bottom corner Note that animations can be run only in slide show mode To run embedded video clips without leaving presentation, select “Enable Content” to allow external content to be played Otherwise, manually copy-paste web links provided in slide note in your browser Andrei V. Nikonov, Ph.D. 1 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Required assignments for Lecture 8 Smart Book 8 – 5 points o Open book policy o 1 attempt  Can check answer once per question  Can save and continue next day  No time limit but must meet the deadline o It will be submitted “As Is” after the deadline DEADLINE For All Submissions – beginning of next lecture class ‒ Deadlines are also given in: o Syllabus o Connect o BrightSpace 7/26/2024 BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 2 Andrei V. Nikonov, Ph.D. 2 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Ch. 17. Applications of Immune Responses Edward Jenner was first to develop the vaccine and use it to immunize humans ‒ In 1796 he had vaccinated a person with vaccinia virus, which led to development of immunity against smallpox infection. Well before him, Chinese were using variolation technique to induce immunity ‒ Material collected from people that were sick with real smallpox was introduced into healthy people 17.1. Principles of immunization Naturally acquired immunity is acquisition of adaptive immunity via natural events Artificially acquired immunity is acquisition of adaptive immunity via human acts Active immunity Passive immunity Microbial antigens appear in the host, Specific antibody introduced in to the host, ‒ Host immune system produces specific antibodies ‒ Host immune system DOES NOT produce specific against the microbe. Long term immunity. antibodies against the microbe. Short term immunity. Infection Antibody transfer from mother to a child ‒ It is result in Naturally Acquired Active Immunity. ‒ It is result in Naturally Acquired Passive Immunity. ‒ A person is running risk of complications or even death ‒ Introduced via placenta and breast-feeding Vaccination Injection of antibody ‒ It is resulting in Artificially Acquired Active immunity ‒ It is resulting in Artificially Acquired Passive immunity ‒ Risks of complications and death are minimal ‒ Antibodies are injected in case of emergency 7/26/2024 Animated VIDEO BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 WWW See slide notes 3 Variolation It was the method of inoculation first used to immunize individuals against smallpox (Variola) with material taken from a patient or a recently variolated individual, in the hope that a mild, but protective, infection would result. Attenuated pathogen – pathogen whose virulence is reduced by its specific cultivation. Elevated temperature used by Robert Koch to grow Vibrio cholerae Naturally acquired immunity – It is resulting from natural processes like: Infection – results in ability of body produce its own specific antibody against a microbe Transfer of antibody from mother to a child – via placenta or via breast milk. It does not result in the ability of child’s body produce its own specific antibody against a microbe Artificially acquired immunity – It is resulting from human acts like: Vaccination – results in ability of body produce its own specific antibody against a microbe Variolation is vaccination against smallpox. It was originally developed by Chinese, then it was improved by Jenner. He called it vaccination. Vaccination is more effective and much safer than variolation. Injection of immunoglobulins produced elsewhere. It does not involve the ability of recipient’s body produce its own specific antibody against a microbe Herd immunity is a phenomenon when at least 85% of a population acquires immunity to a disease either through natural immunity or a vaccination. It prevents major outbreaks of the contagious disease as the rest of population is also protected due to interruption of disease transmission. Individual immunity is when a particular individual has immunity to a disease. It protects the individual from getting a disease Active immunity means that human body is producing its own antibody against microbe or toxin and has the antibody in blood stream. Passive immunity means that human body is not producing its own antibody against microbe or toxin. The antibody present in blood stream were produced elsewhere. Andrei V. Nikonov, Ph.D. 3 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 18.2. Vaccines and immunization procedures Vaccine is preparation of a pathogen or its parts used to induce active immunity Herd immunity is achieved when critical portion of population develop resistance to the disease Attenuated and inactivate vaccines Characteristics Attenuated vaccine Inactivated vaccine Microbe Weakened but alive. It can grow in host Killed. It can not grow in the host Required doses Single Multiple Need for adjuvant (i.e. alum) No Yes Route of administration Injection; Oral or nasal drops Injection only Risk to healthy individuals Very low None Risk to immuno-compromised or fetus Can be significant None Duration of immunity Long term Short term Stability of vaccine at room temperature Poor Good (except for mRNA vaccines) 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 WWW See slide notes 4 Adjuvant It is a substance that increases the immune response to antigen. Alum is most common adjuvant used. Immunization (vaccination) It protects via individual immunity & also herd immunity (when 85% or more of population is immunized). Attenuated vaccines Attenuated vaccine is the weakened but still alive pathogen that can grow. In healthy individuals, it can not cause a disease. However, It can cause a disease in individuals with compromised immune system One injection is usually sufficient to develop strong immunity Examples: vaccines against most viral infections. Polio (Sabin); Measles, Mumps, Rubella (MMR); Chickenpox (Varicella-Zoster) Smallpox (Vaccinia virus) Inactivated vaccines Inactivated vaccine is a killed pathogen or its parts. It can not grow or cause a disease in healthy or immunocompromised individuals. Multiple injections are required with adjuvant to develop strong immunity Examples: Against dangerous or latent viruses: Polio (Salk), Hepatitis, Rabies Against dangerous bacterial infections: Diphtheria, Tetanus, Pertussis (DTaP); Meningitis (MCN-4, MPSV-4, Hib); Pneumonia (PCV-7, PCV-13) Vaccines available in the USA by disease https://www.vaccines.gov/diseases/index.html Andrei V. Nikonov, Ph.D. 4 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Adjuvant (the substance that help to induce a better immune response) Types of inactivated vaccines must be used with inactivated vaccines. Most common is Alum (salts of Al) Inactivated whole agent vaccines Subunit vaccines Virus-like particle (VLP) vaccines They work by delivering foreign DNA to human Toxoids cell to make the microbial protein antigen It would trigger immune response leading to antibody production that would afford the protection against the disease Polysaccharide vaccines It is used in making Russian vaccine against COVID-19, Sputnik V Conjugated vaccines Peptide vaccines Synthetic fragments of antigen Stable to heat, Have little unwanted material Recombinant vaccines Cause minimal side effects 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 WWW See slide notes 5 Types of inactivated vaccines It also includes adjuvants – the substances that help to induce a better immune response. The most common adjuvant used is Alum – precipitated aluminum salts. Inactivated whole agent vaccines contain bacteria or viruses treated with formalin that kills or inactivated microbes by cross-linking their proteins – EXAMPLES: Salk polio vaccine, Influenza vaccine, Rabies vaccine, Hepatitis A It protects only against one disease Subunit vaccines contain purified from pathogen key protein antigens or antigenic fragments – EXAMPLES: Acellular Pertussis vaccine (it is a part of DTaP vaccine), Toxoids – toxins purified from pathogen that are inactivated but retaining antigenic properties of native toxins. EXAMPLES: Diphtheria and Tetanus toxoids that are part of DTaP vaccine Polysaccharide vaccines contain polysaccharides purified from capsules. They can not be used in immunization of young children, reserved for immunization of adults only. EXAMPLES: PPSV (pneumococcal polysaccharide vaccine) against Streptococcus pneumoniae Conjugated vaccines contain polysaccharides linked to proteins, which turns polysaccharides into T-cell dependent antigens. EXAMPLES: Hib vaccine (against Haemophilus influenza serotype B), PCV-7, PCV-13 (pneumococcal conjugated vaccine against 7 or 13 antigens against Streptococcus pneumoniae) It can protect against several diseases. Recombinant vaccines produced by genetically modified microorganisms – EXAMPLES: Hepatitis B vaccine is produced in yeasts producing part of viral coat proteins Vaccines available in the USA by types https://www.vaccines.gov/basics/types/index.html Q. Why are polysaccharide vaccines cannot be used in young children? Andrei V. Nikonov, Ph.D. 5 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Vaccination uses and benefits Disease US annual cases before Decrease after immunization Vaccine is approved Measles cases Number of reported cases immunization reported in the USA Smallpox 48,164 (1900-1904) Eradicated -- 350,000 Diphtheria 175,885 (1920-1922) Only imported cases Measles 503,282 (1958-1962) Until recently only imported Polio 16,316 (1951-1954) Until recently only imported Mumps 152,209 (1968) 99.8% Tetanus 1,314 (1922-1926) 98.1% Incidence of influenza vs. Doses of vaccines, mill. (bars) Incidence per 100,000 (line) Rubella 50,230 (1966-1969) 98% vaccination in the USA Pertussis 147,271 (1922-1925) 93.4% Smallpox eradication: Watch optional video posted on blackboard Jonas Salk (1914 - 1995) has developed IPV 7/26/2024 VIDEO BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 6 Smallpox [EXTRA] It is an acute infectious and contagious disease of humans, caused by either of two virus variants named Variola major and Variola minor. Variola is a derivative of the Latin varius, meaning “spotted”, or varus, meaning "pimple". Smallpox localizes in small blood vessels of the skin and in the mouth and throat. In the skin, this leads to characteristic rash, and later to pus-filled pustules mainly localized on limbs and head. Variola major produces a more serious disease and has an overall mortality rate of 30– 35%, while Variola minor causes a milder form of disease with mortality rate about 1% of infected. Vaccine against smallpox is based on Vaccinia virus, the microbe that infects the cows causing cowpox but it does not cause disease in humans. Vaccinia virus is closely related to Variola virus and it has proteins with antigenic properties similar to Variola virus Therefore, antibody produced against Vaccinia virus are also protective against smallpox. Last outbreak in Europe was in Yugoslavia in 1972 Last naturally occurring case was in Somalia in 1977 Global eradication was certified on May 8th, 1980 Video [EXTRA] Eradication of smallpox https://youtu.be/dVmkYSkQEN8 Andrei V. Nikonov, Ph.D. 6 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Unvaccinated versus vaccinated Unvaccinated Vaccinated Severe disease Moderate or Mild disease Moderate disease Asymptomatic infection Moderate disease Asymptomatic infection Mild disease Not infected Mild disease Not infected Mild disease Not infected Mild disease Not infected Asymptomatic infection Not infected Asymptomatic infection Not infected Asymptomatic infection Not infected 7/26/2024 BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 7 Andrei V. Nikonov, Ph.D. 7 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Effect of PCV-7and PCV-13 on prevalence of invasive pneumococcal (caused by Streptococcus pneumoniae) disease in the USA among young children and elderly PCV-7 vaccine is pneumococcal conjugated vaccine against 7 antigens that was approved in 1997 for immunization of children and adults. PCV-13 vaccine is pneumococcal conjugated vaccine against 13 antigens that was approved in 2009 for immunization of adults only. 7/26/2024 BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 8 PCV-7 vaccine It is pneumococcal conjugated vaccine against 7 antigens that was approved n 1997 for immunization of children and adults. Its introduction resulted in 4-fold decline in prevalence (# cases reported for 100,000 people) of invasive pneumococcal disease among young children. The use of this vaccine in immunization of adults resulted in 30% decline in prevalence of invasive pneumococcal disease among old adults PCV-13 vaccine It is pneumococcal conjugated vaccine against 13 antigens that was approved in 2009 for immunization of adults only. Replacement of PCV-7 for PCV-13 in immunization of adults led to decline in prevalence of invasive pneumococcal disease not only adults but also among young children who were still immunized by PCV-7 Q. PCV13 vaccine is conjugated vaccine and triggers immune response in young children. WHAT might be the reasons for not using PCV-13 vaccine in immunization of young children? Q. PCV-13 is used in immunization of adults only. WHY the use of PCV-13 had reduced the prevalence of pneumococcal disease among young children as well? Andrei V. Nikonov, Ph.D. 8 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Routine vaccines The USA principal vaccines ‒ Preventing bacterial diseases o DTaP, replaced older DTP or DTwP. Contains purified diphtheria & tetanus toxoids, fragments of Bordetella pertussis cell wall o MCV-4 or MPSV-4. Contain purified polysaccharide from Neisseria meningitidis o PCV-7, PCV-13, PCV-23. Contains polysaccharides of 7, 13, or 23 serotypes of CV – conjugated Streptococcus pneumoniae conjugated to vaccine; diphtheria toxoid, CRM197 PSV – o Hib. Contains polysaccharides of Hemophilus polysaccharide influenza type b conjugated to a carrier protein vaccine ‒ Preventing viral diseases o MMR. Contains attenuated viruses of Measles, Mumps, Rubella o Varicella-Zoster (Chickenpox). Contains attenuated virus of Herpes-Zoster o Salk (Sabin) polio vaccine. Contains inactivated (attenuated) poliomyelitis virus o HDCV. Contains inactivated rabies virus 2019 7/26/2024 BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 9 DTaP vaccine DTaP is inactivated subunit vaccine that contains diphtheria and tetanus toxoids and acellular component of Bordetella pertussis. Previous vaccine, DTwP, had the whole killed cells of B. pertussis. DTaP vaccine provides protection against diphtheria, tetanus, and pertussis (whooping cough). It can be used in pregnant women and immunocompromised. Q. WHAT was the reason in replacement of DTP or DTwP vaccine with DTaP? MMR vaccine MMR contains three attenuated live viruses: measles, mumps, and rubella and provides protection against corresponding diseases. The vaccine can not be used in pregnant women due to high risk of rubella congenital syndrome. MCV-4and MPSV-4 vaccines Immunization with these vaccines provides protection against meningitis caused by Neisseria meningitidis MCV-4 – conjugated vaccines against 4 antigens. Approved for use in young children. MPSV-4 – polysaccharide vaccine against 4 antigens. Reserved for use in adults Conjugation of bacterial polysaccharides with CRM197 CRM197 is non-toxic genetically modified protein that increases efficiency of immune response to bacterial polysaccharide present in vaccine and conjugated to CRM197 Andrei V. Nikonov, Ph.D. 9 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 An example of vaccination strategy – The campaign to eliminate poliomyelitis 100,000 Salk inactivated vaccine Paralytic polio cases Poliomyelitis (Polio) (IPV) -- 1954 reported in the USA ‒ Acute viral disease transmitted via oral-fecal route 10,000 o Up to 95% - asymptomatic; Up to 8% - mild disease; Sabin attenuated vaccine (OPV) -- 1963 o Up to 2% - meningitis 1,000 o Up to 0.5% - paralytic cases 1980: Last indigenous case reported in USA 100 10 1 1950 1954 1958 1962 1966 1970 1974 1978 1982 1986 1990 1994 1998 2002 2006 2010 Paralytic polio cases reported globally Salk inactivated vaccine (IPV) 100,000 ‒ Contains all 3 types of inactivated viruses ‒ Disadvantage – requires multiple injections, no IgA 10,000 produced => virus can replicate in guts of vaccinated 1,000 Sabin attenuated vaccine (OPV) ‒ Contains viral strains that replicate in intestines 100 India ‒ Advantages – requires only one oral administration, Russia Tajikistan produces better mucosal immunity (IgA) => better 10 herd immunity 1 ‒ Disadvantage – virus can revert to pathogenic strain 1980 1983 1986 1989 1992 1995 1998 2001 2004 2007 2010 o One case of polio in ~2.4 million immunized 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 10 Poliomyelitis as an example of importance of immunization Before the vaccines, ~50,000 polio paralytic cases were reported in the USA annually Vaccination of public with Salk inactivated polio vaccine (IPV) in 1954 had reduced number of paralytic polio cases to under 600 in 1962. NOTE: Salk polio vaccine protects only against paralytic polio cases. Intestines of immunized by Salk inactivated vaccine (IPV) still can be infected by the virus allowing it to spread in population. Introduction of Sabin attenuated vaccine (OPV) in 1963 further reduced number of paralytic polio cases to 5 in 1973. After 1998, there were no paralytic polio cases reported in the USA. However, the outbreak of polio in former USSR in 1991-1996 due to the lapse in immunization of general public, indicates that the disease is under control only if there is a herd immunity. NOTE: Polio was re-discovered in the USA in 2022 Outbreak of poliomyelitis in former USSR in 1991-1996 This outbreak of polio indicate that the disease still has ability to return. The outbreaks had occur due to inefficient service in public health (particularly lapse in immunization of infants) on territory of former USSR after breakup. The magnitude of paralytic polio outbreak was limited to 10-110 cases reported annually in Russia or Tajikistan alone with no cases reported before The outbreak did not reach larger proportions as the population still had herd immunity against polio due to stringent immunization efforts in previous years. Andrei V. Nikonov, Ph.D. 10 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Study of 2019: Americans with higher education and with higher income more likely to see high benefits and low risks of vaccines New vaccines required Disease Estimated impact Malaria Up to 500,000,000 infected. Up to 3,000,000 deaths globally / year Hepatitis C 170 million infected globally Cancer 1 in 3 in the US may get cancer Genital herpes 45 million infected in the USA Strep throat 20 million cases in the USA /year Influenza ~50 million infected globally / year NIV/AIDS ~37 million infected globally 7/26/2024 BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 11 Vaccine against cancer It is practically impossible to develop universal vaccine against all types of cancer Each type of cancer caused by different factors and affects specific type of cell, tissue. Vaccine against herpes Vaccine can prevent new infections However, if infection is established it can not be cured. Q. If person is infected with herpes virus, that person would carry the virus for the rest of life. WHY? Vaccine against strep throat Strep throat is pharyngitis caused by Streptococcus pyogenes Development of effective vaccine against strep throat is very difficult as there are over 80 known serotypes of Streptococcus pyogenes Any confirmed strep throat case must be promptly treated with antibiotics If strep throat is left untreated, there is a great chance of serious complications. Vaccine against influenza (flu) New vaccine is required due to high mutation rate of influenza virus Q. Why is influenza virus has very high mutation rate? Vaccine against HIV No vaccines effective against HIV are developed yet. Andrei V. Nikonov, Ph.D. 11 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 17.4. Principles of immunoassays Immunoassay is any in vitro test that takes advantage of specific antigen-antibody interaction with formation of immune complex to detect or quantify an antigen or antibody in the sample. ‒ Detection answers “Yes” / “No” to question “is antigen or antibody present in sample?” + = ‒ Quantification determines the amount of antigen or antibody present in the sample. Ab Ag I.C. Quantifying antigen-antibody reactions The concentration of antibody is determined by making serial dilutions which are mixed with the antigen ‒ The last dilution of antibody at which antigen-antibody reaction can be detected defines the titer (concentration) of antibody ‒ Titer of antibody is expressed as inverse to that dilution – antibody titer 1:128 means that if the antibody is diluted 128 times, the rection between antibody and antigen will be detected. Two-fold dilutions of serum are added to the wells containing same amount of antigen: 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 NOTE: the same antibody + + + + + + + – would have higher or lower titer for more or less sensitive methods Antibody titer is 1:128 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 12 In patient, the presence of specific antibody is determined in serum or in plasma: Serum – fluid part remaining after removal of both, the blood cells & formed blood clot. ‒ It is the most common source for specific antibody. Therefore, all methods involving the use of specific methods are also called serological methods Plasma – fluid part of blood after removal of blood cells only. ‒ It is treated with anticoagulants to prevent blood clot formation. Sero-conversion is referring to appearance of specific antibody in the blood due to immune response. A host’s status is converted from sero-negative to sero-positive Andrei V. Nikonov, Ph.D. 12 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Obtaining known [specific] antibody Polyclonal antibodies (Pabs) Monoclonal antibodies (Mabs) Antigen is injected into laboratory animal or Antigen is injected into mouse to protocol volunteer several times, according to Spleen is collected and cells are extracted from it protocol The cells are mixed with myeloma cells and forced to fuse The blood is collected and serum is The cells are plated on multiwell plates – 1 cell / well and are separated from blood cells grown in selective medium – not fused cells are eliminated Resulting antiserum contains specific Pabs The growing clones are tested for Mabs. Each of them is: that are: ‒ Produced by plasma cells derived from single naïve B cell ‒ Produced by plasma cells derived from ‒ Monovalent – can recognize single epitope of the antigen various naïve B cells ‒ Produce weaker signal but less likely to be cross-reactive ‒ Polyvalent – can recognize various epitopes of the same antigen - produce much stronger signals ‒ More likely to be cross-reactive Production of monoclonal antibodies ‒ Watch the video posted on blackboard – 1:31 7/26/2024 Animated VIDEO BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 13 Properties of polyclonal antibody They are called polyclonal as they are mixture of antibodies produced by different clones of plasma cells descended from various naïve B-cells. These antibodies can recognize various epitopes on the same antigen Therefore, are more likely cross-react with other antigens. Polyclonal antibodies are produced by injecting antigen into volunteers or lab animals. Vacutainer It is a device used to collect the blood and isolate antiserum It is a test tube with negative pressure and closed with rubber stopper. The test tube has a gel that allows blood cells to go through when test tube is subjected to centrifugation, thus separating the antibody-containing liquid phase (plasma) of blood from blood cells. Properties of monoclonal antibody They are called monoclonal as they contain the antibody produced by single clone of plasma cells descended from single naïve B-cell. These antibodies can recognize only single epitope Therefore, they are less likely to cross-react with other antigens, including human cells and tissues. Video Production of monoclonal antibodies 1:31 https://youtu.be/3kfFXdZ9Gk8 Andrei V. Nikonov, Ph.D. 13 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Genetically modified Mabs used in medical applications EXAMPLES: ‒ Omalizumab is a Mouse antibody Human antibody humanized Mab that is Constant region of Constant region of being used to treat Type antibody is mouse-specific antibody is human-specific I allergy reactions. It The antibody is causing The antibody is not binds the Fc portion of immune response in causing immune response IgE, preventing it from humans in humans binding to mast cells Monoclonal antibodies can Monoclonal antibodies can and basophils. be produced but they can not be produced ‒ Leronlimab (PRO 140) be injected only once in a humanized Mab that patient binds to CCR5 co- receptor thus preventing entry of HIV into host Chimerical antibody Humanized antibody cells. Constant region of mouse Constant and some ‒ Antibody-Drug antibody is replaced with variable regions of mouse conjugates ones of human antibody antibody are replaced (immunotoxins) Immune response to with human ones these antibodies is muted There is no immune response to these What are antibody- in humans Monoclonal antibodies can antibodies in humans drug conjugates? be produced and can be Monoclonal antibodies can Watch the video injected more than once be produced and injected uploaded on multiple times blackboard 7/26/2024 Animated VIDEO BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 14 Chimerical antibody versus humanized antibody Chimerical antibody is MAb produced in hybridomas derived from genetically modified mice where mouse constant region of antibody is replaced with human one. It causes muted immune response and can be injected into patient more than once. Humanized antibody is MAB produced in hybridomas derived from GMO mice where mouse constant and some of variable region of antibody are replaced with the human ones. Humanized antibody does not trigger immune response in humans and, therefore, better suited for medical use. Some of them have been approved for the use as immunotoxins in cancer therapy.. EXAMPLES: Omalizumab is a recombinant humanized monoclonal antibody that is used to treat Type I allergy reactions. This IgG antibody works by binding to the Fc portion of IgE antibodies, preventing them from binding to mast cells and basophils, and therefore, preventing. Leronlimab (PRO 140) is recombinant humanized monoclonal antibody directed against CCR5 that is used to prevent entry of HIV in T helper cells Antibody-Drug conjugates (Immunotoxins) They are antibodies conjugated with toxin. They specifically binds to a particular type of cell (i.e. cancer cell) Then, it is taken inside the cell (internalized) and toxin kills the targeted cell. Humanized antibodies are particularly well suited for use as immunotoxins since they do not trigger immune response in humans. So far, 9 monoclonal antibodies have been approved for cancer therapy Video What is antibody-drug conjugates? 3:32 https://youtu.be/GD0gcZoqtcM Andrei V. Nikonov, Ph.D. 14 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 17.5. Common types of immunoassays Antibodies are protein molecules, that can not be observed by using conventional microscopy methods ‒ Therefore, many assays were developed to observe and monitor the antigen-antibody interactions. They are two types: 1. Immunoassays that use labeled antibodies 2. Immunoassays that involve formation of visible antigen-antibody aggregates Immunoassays that use labeled antibodies Direct Immunoassays Indirect Immunoassays ‒ Specific antibody is directly labeled by a tag ‒ Specific antibody remains untagged. ‒ The tag may be – enzyme; colloidal gold; radioactive ‒ SECONDARY antibody is labeled by a tag material; fluorescent dye; Tagged secondary antibody Specific Specific antibody Y Step 1. Labeling Tagged specific antibody directed against specific antibody antibody Step 2. Step 2. Step 1. Immunoassay Relatively: Relatively: Y ‒ More specific ‒ Less specific ‒ More expensive ‒ Less expensive ‒ Low signal Sample with antigens ‒ High signal Sample with antigens 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 15 Direct immunoassays They are particularly useful in IDing of unknown antigen in the sample Advantages Reduced risk of cross-reaction Disadvantages: Each specific antibody must be labeled – time consuming and expensive Reduced intensity of the signal. Indirect immunoassays They are particularly useful in detection in the sample the antibodies against known antigen Advantages Each specific antibody does not need to be labeled – just buy commercially available secondary antibodies – time saving and less expensive. Increased intensity of the signal as more than one secondary antibody molecule can bind to specific antibody Disadvantages Increased risk of cross-reactivity of secondary antibody. Each secondary antibody must be tested for its cross-reactivity Andrei V. Nikonov, Ph.D. 15 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 It also can be direct or indirect, depending on what antibody is labeled Fluorescent antibody test ‒ Indirect test is more preferable Advantages: Specific Enables detection and IDing of microbes directly in Fluorescein antibody the sample, including those that can not be cultivated in the laboratories. Faster than bacterial cultivation. + Disadvantages: Labor intensive & time consuming as it requires Sample with a bacterium individual preparation of each sample and individual carrying the antigen examination of each slide under fluorescent microscope Tagged specific antibody Sample stained n fluorescent antibody test is + visualized under fluorescence microscope. Labeled bacterium 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 16 Andrei V. Nikonov, Ph.D. 16 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Enzyme-linked immunosorbent assay (ELISA) To detect the presence of antigen, the enzyme-labeled antibody is used in ELISA, which converts the color-less substrate into a colored product ‒ Advantages: relatively simple and easy to do; highly sensitive; very fast results ‒ Disadvantages: false-positive results are quite common. Therefore, the ELISA results always must be confirmed by other methods Direct ELISA Indirect ELISA Direct sandwich Indirect sandwich It can be used to detect in sample the antigen only It can be used to detect in sample the antigen and antibody ELISA – Ag and Ab detection ELISA – Ag and Ab detection Y Y Y Y Y Y – antigen Y – specific antibody directed against antigen – colored product * – enzyme Y – secondary antibody directed against primary antibody. Y – capture antibody Multiple secondary Abs can bind to same primary Ab 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 17 The plastic used to make multi-well plates used in ELISA is positively charged. -- therefore, it is readily binding proteins or other negatively charged molecules After coating the well with antigen, the remaining binding sites of the plastic must be blocked -- inert proteins like ovalbumin or weak detergents like Tween 20 can be used as blocking agents -- blocking of binding sites prevents unspecific binding of specific antibodies to the plastic and thus increases the specificity of the signal To ensure appropriate orientation of capture antibody in sandwich ELISA, the wells are coated with protein A or protein G first -- then after, a well is coated with capture antibody, followed by blocking solution. Andrei V. Nikonov, Ph.D. 17 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Western blotting It is a method of detecting the presence of protein antigen (must be loaded on the gel) and establishing its molecular weight, or antibody (must be used as primary antibody) in the sample It is done in two steps: 1) SDS gel electrophoresis 2) Western blotting Separation of proteins in the gel a) Transfer of proteins b) Detection of the protein c) Visualization of the protein on membrane labeled 1 2 3 4 5 1 2 3 4 5 sponge Y secondary filter paper antibody membrane Y primary gel antibody filter paper proteins membrane sponge membrane More details on SDS gel electrophoresis and Western blotting? Watch optional videos posted on blackboard 7/26/2024 Animated VIDEO BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 18 Videos: SDS-PAGE 8:37 https://youtu.be/i_6y6Z5UvwE Western blotting 10:55 https://youtu.be/Ll_7z4YS2Ak Andrei V. Nikonov, Ph.D. 18 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Immunoassays that involve visible antigen-antibody aggregates These methods were extensively used in the past ‒ but now they have been largely replaced by more sensitive assays that use labeled antibodies Agglutination reactions Precipitation reactions In these reactions the cells are cross-linked by In these reactions the soluble proteins are cross- antibody forming large aggregates linked by antibody forming visible precipitates They can be used to detect the presence of They can be used to detect the presence of ‒ Specific viruses ‒ Specific soluble antigen ‒ Specific antibodies ‒ Specific antibodies ‒ Measure the antibody in the serum ‒ Measure the antibody in the serum Examples: Examples: ‒ Latex agglutination reaction ‒ Ouchterlony assay ‒ Serum agglutination assay ‒ Blood type agglutination test More details on these reactions, assays and tests? Watch optional videos uploaded on blackboard 7/26/2024 VIDEO BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 19 [EXTRAS] Videos: Agglutination reactions Latex agglutination reaction 2:41 https://youtu.be/oXL1rH11MTg Measuring Serum Antibody with an Agglutination Assay 4:07 https://youtu.be/aV8NPSiPh_4 What Are Blood Types? - Agglutination Test 3:00 https://youtu.be/Zsdhe0xFBf4 Precipitation reactions Ouchterlony Assay 3:46 https://youtu.be/Fnx5CkGRBEM Andrei V. Nikonov, Ph.D. 19 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Ch. 20. Antimicrobial Medications Selective toxicity is the most important characteristic of any antimicrobial medication – It is ability of antimicrobial drug to stop the grown or kill a microbe without causing damage to the host (human) 20.1. History and development of antimicrobial medications Antimicrobial medication [drug] is any compound that can stop the growth or kill a microbe – Antibiotic is antimicrobial drug produced by bacteria or fungi. So, some of antimicrobial drugs are not antibiotics. Discovery of antimicrobial medications Salvarsan (discovered in 1910) Prontosil (discovered in 1931) The first synthetic (arsenic) compound used as When this red dye is metabolized in blood, it splits antimicrobial medication forming sulfanilamide – It is effective against Treponema pallidum – It is effective against Streptococcus species 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 20 Andrei V. Nikonov, Ph.D. 20 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Discovery of antibiotics Penicillin (discovered in 1928) Streptomycin (discovered in 1943) Isolated from mold Penicillium natatum by Alexander Isolated from actinobacteria Streptomyces griseus by Fleming (Scotland) in 1928. Selman Waksman (Rutgers Univ., USA) in 1943 – Purified by Florey and Chain. Inhibits protein synthesis. – Nobel prize in medicine for discovery of penicillin was – Bactericidal, awarded to all three in 1945. – Broad-spectrum (effective against Gram-positive and Inhibits cell wall synthesis. Gram-negative bacteria – Bactericidal, – It is used in treatment of tuberculosis. – Narrow-spectrum (effective against Gram-positive bacteria Normal growth of Staphylococcus aureus Zone of inhibition Colony of Penicillium notatum 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 21 Penicillin It was discovered by Alexander Fleming (Scotland) in 1928, while he was working with pure cultures of Staphylococcus aureus. One day he noticed that bacterial growth n Petri dish was partially suppressed in close proximity to colonies of a mold contaminant, Penicillium notatum (chrysogenum). In 1945, he shared Nobel Prize with Florey and Chain, who purified the drug. Antibiotic inhibits the synthesis of bacterial cell wall and is effective mainly against Gram positive bacteria. It is also effective against Neisseria, Gram negative bacteria. Natural penicillins are isolated from fungi: Penicillin G is sensitive to acidic environment of stomach and, therefore, must be administered intramuscularly. Penicillin V, is acid-resistant and, therefore, can be taken orally. Semi-synthetic penicillins are isolated from fungi and then chemically modified that led to new properties of penicillins: Ampicillin has broader antimicrobial spectrum Methicillin is resistant to inactivation by penicillinase, bacterial enzyme that can digest natural penicillins. Streptomycin It was discovered by Selman Waksman (USA) in 1943, while he was working at Rutgers University with bacterial flora of soil. Antibiotic was isolated from actinobacterium Streptomyces griseus found in soil. Antibiotic irreversibly inhibits protein synthesis. It is antibiotic of choice in treatment of tuberculosis. Andrei V. Nikonov, Ph.D. 21 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Development of new antimicrobial medications Most of the antibiotics are coming from microbial species living in the soil In the 1960-ties, it was discovered that chemical modification of antibiotic’s structure improve it’s properties ‒ Natural antibiotics are the ones isolated from microbes – Penicillin G ‒ Semi-synthetic antibiotics are the ones with chemically modified structures – Methicillin, Ampicillin Sources of antibiotics Group of microbes Species Antibiotic Target Spectrum / Action Fungi Cephalosporium spp. Cephalosporin Cell wall synthesis Broad / Bactericidal Penicillium natatum Penicillin Cell wall synthesis Narrow (G +) / Bactericidal Actinobacteria Streptomyces aureofaciens Tetracycline Protein synthesis Broad / Bacteriostatic Streptomyces cattleya Carbapenem Cell wall synthesis Broad / Bactericidal Streptomyces erythraeus Erythromycin Protein synthesis Narrow (G +) / Bacteriostatic Streptomyces griseus Streptomycin Protein synthesis Broad / Bactericidal Streptomyces venezuelae Chloramphenicol Protein synthesis Broad / Bacteriostatic Firmicutes Bacillus lichenformis Bacitracin (*toxic) Cell wall synthesis Narrow (G +) / Bactericidal Paenibacillus polymyxa Polymyxin (*toxic) Membrane integrity Narrow (G – ) / Bactericidal 7/26/2024 BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 WWW See slide notes 22 [EXTRA] List of antibiotics on Wikipedia https://en.wikipedia.org/wiki/List_of_antibiotics Fungi Penicillins are inhibiting the synthesis of bacterial cell wall. Diverse family of antibiotics. Penicillins are more effective against Gram-positive bacteria and are less effective against Gram-negative bacteria, as it can not penetrate their outer membrane. However, it can have effect only on actively growing microbes. Cephalosporins are inhibiting the synthesis of bacterial cell wall. However, it can have effect only on actively growing microbes. Cephalosporins are effective against both Gram-positive and Gram-negative bacteria, as it can cross outer membrane of later bacteria. However, they are less effective on Gram- positive bacteria as tend to have low affinity to penicillin-binding proteins of these bacteria Actinobacteria Chloramphenicol is reversibly inhibiting protein synthesis. Bacteriostatic. Effective against both Gram- positive and Gram-negative bacteria. It is used as last resort antibiotic for life-threatening infections due to its high toxicity to humans. Erythromycin is reversibly inhibiting protein synthesis. Bacteriostatic. Effective against both Gram- positive and Gram-negative bacteria. Streptomycin is IRREVERSIBLY inhibiting protein synthesis, effectively killing microbes. BACTERICIDAL. Effective against both Gram-positive and Gram-negative bacteria. One of the antibiotics used in combination with others in treatment of tuberculosis. Tetracycline is reversibly inhibiting protein synthesis. Bacteriostatic. Effective against both Gram positive and Gram-negative bacteria. Firmicutes Bacitracin is inhibiting synthesis of bacterial cell wall. It is effective against Gram positive bacteria. High toxicity limits the use to topical applications. Polymyxin is causing damage to membrane. Effective against Gram negative bacteria only Andrei V. Nikonov, Ph.D. 22 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 20.2. Characteristics of antimicrobial medications It should cause hard to microbes, not to the human host. It is expressed as Selective toxicity ‒ therapeutic index = [lowest dose toxic to humans] / [lowest dose toxic to microbe] Bacteriostatic antimicrobial inhibits the growth of a microbe. Antimicrobial action Bactericidal antimicrobial kills the microbe Target in the cell – cell wall, ribosomes (protein synthesis) etc Broad-spectrum drugs affect wide range of bacteria, “G +” and “G –” Spectrum of activity Narrow-spectrum drugs affect limited range of bacteria, “G +” or “G –” Antagonistic effect – combination makes outcome worse Effects of combination Synergistic effect – combination makes outcome better Tissue distribution (few cross into cerebrospinal fluid) Pharmacokinetics Metabolism and Excretion define the drug’s the half-life Allergic reactions and Toxic effects Adverse effects Suppression of normal microbiota (flora) Intrinsic (innate) resistance to the drug Resistance to antimicrobials Acquired resistance to the drug 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 23 Antimicrobial action It includes whether the drug kills microbe or just inhibit the growth. It also includes the drug’s targets in the microbial cell. Bacteriostatic antimicrobial medication Bactericidal antimicrobial medication Drug targets in microbe – Cell wall; Protein synthesis (ribosomes); DNA synthesis (DNA polymerase) Effect of combination of antimicrobial medications Synergism occurs when the effect of two drugs together is greater than the effect of either alone. EXAMPLE: Ethambutol + Isoniazid + Streptomycin in TB treatment. The first two reduce hydrophobicity of cell wall of M. tuberculosis by inhibiting the synthesis of mycolic acid, while the third drug irreversibly binds to ribosome and inhibits the protein synthesis in the cell. EXAMPLE: Sulfa drug and trimethoprim are inhibiting two enzymes of folic acid biosynthesis metabolic pathway. Bacteria is unlikely to develop resistance against both antibiotics at the same time Antagonism occurs when the effect of two drugs combined is less than an effect of either drug used alone. EXAMPLE: Penicillin and chloramphenicol combined have bacteriostatic effect, while penicillin alone kills bacteria Adverse effects It includes possible side-effects resulting for antimicrobial medication use. EXAMPLES: Allergic reactions – allergy to penicillin (anaphylaxis – see lecture 9) Toxic effects – chloramphenicol may cause fatal aplastic anemia, a condition when body can not produce blood cells Suppression of normal flora – Antibiotic-associated colitis caused by Clostridium difficile Andrei V. Nikonov, Ph.D. 23 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 20.3. Mechanisms of actions of antibacterial medications In bacterial cell, antibacterial medications can target: ‒ Cell wall synthesis; Protein synthesis; Nucleic acid synthesis; Metabolic pathways; Cell membrane integrity Inhibition of cell wall synthesis -lactam antibiotics Glycopeptide antibiotics Bacitracin ‒ They compete with side chain for ‒ Vancomycin competes with PBP ‒ It inhibits the export of NAM- active site of PBP for binding to peptide side chain NAG out of the cell Penicillin Vancomycin NAM-NAG polymer binding Plasma membrane Cell wall Penicillin protein binding Vancomycin binds to protein peptide side chain of peptidoglycan X NAM-NAG produced in Peptide side chain cytoplasm X Cross-link is formed If bacitracin present If vancomycin is present, no cross-links are Site of cell wall synthesis is outside X formed + P P Penicillin Vancomycin can not cross outer membrane of Gram negative bacteria Bacitracin is toxic to humans as it disturbs plasma membrane of human cells 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 24 Beta-lactam drugs They include penicillins and cephalosporins that are inhibiting the synthesis of bacterial cell wall by binding to active site of penicillin-binding protein and thus preventing the cross-linking of peptidoglycan polymeric fibers. Bacterial resistance to beta-lactams – 1) mutation in penicillin-binding protein so that it no longer binds penicillin but it is still can cross-link peptidoglycan fibers; 2) microbe produces enzyme (penicillases or b-lactamases that break down the b-lactam ring of penicillins or cephalosporins. Natural penicillins are narrow spectrum antibiotics, while semi-synthetic penicillins and cephalosporins are broad spectrum antibiotics. It can be administered intravenously or orally. Vancomycin It is inhibiting the synthesis of bacterial cell wall by binding to side chain of NAM-NAG and thus preventing cross-linking of peptidoglycan polymeric fibers. Bacterial resistance to vancomycin – mutation in pentapeptide side chain of NAM-NAG that replaces the terminal alanine (Ala) with lactic acid, so that it no longer binds vancomycin but it is still can be cross-linked by penicillin binding protein. It is narrow spectrum antibiotic as it can not cross outer membrane of Gram negative bacteria. and, therefore, it can be used against Gram positive bacteria only. It must be administered only intravenously due to it’s poor absorption from intestinal tract Bacitracin It is inhibiting the transport of peptidoglycan precursors across membrane, from cytoplasm to site of cell wall synthesis It inhibits cell wall synthesis by interfering with transport of peptidoglycan precursors across plasma membrane. The drug inhibits de-phosphorylation of bactoprenol-phosphate that is a carrier for the precursors. However, due to high toxicity, it’s use is limited to topical applications. It is a common ingredient in non- prescription first-aid ointments. Andrei V. Nikonov, Ph.D. 24 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Inhibition of protein synthesis Aminoglycosides Tetracyclines ‒ Streptomycin is ‒ Tetracycline is o Irreversibly binding to 30S ribosomal subunit o Reversibly binding to 30S ribosomal subunit  Blocks initiation of translation and causes  Blocks attachment of tRNA to ribosome thus misreading of translated mRNA preventing translation from continuing. o Bactericidal o Bacteriostatic o Broad-spectrum o Broad-spectrum  Effective against bacteria capable of cellular  Effective against any bacteria, Gram- respiration, Gram-positive or Gram-negative positive or Gram-negative o Side effects: hearing loss, kidney damage o Side effects: “tetracycline smile”, tinnitus Macrolides Chloramphenicol ‒ Erythromycin is ‒ Chloramphenicol is o Reversibly binding to 50S ribosomal subunit o Reversibly binding to 50S ribosomal subunit  Blocks translocation of ribosome thus  Blocks peptide bond formation thus preventing translation from continuing. preventing translation from continuing. o Bacteriostatic o Bacteriostatic o Narrow-spectrum o Broad-spectrum  Effective against Gram-positive bacteria,  Effective against any bacteria, Gram- including Mycoplasma spp. positive or Gram-negative o Side effects: diarrhea, psychotic reactions o Side effects: aplastic anemia – thus limited use. 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 25 Streptomycin Class – aminoglycosides. EXAMPLES: of other antibiotics from this class are Gentamicin, Kanamycin It is bactericidal antibiotic as it irreversibly binding to 30S ribosomal subunit and inhibits initiation of translation. It is broad-spectrum antibiotic that is effective against Gram-positive, Gram-negative and Acid-fast bacteria. However, it is not effective against Enterococcus and Streptococcus. Side effects: Extended use may lead to nephrotoxicity. Bacterial resistance: Microbes can produce enzymes that chemically modifies the antibiotic. Tetracycline Class – tetracyclines. EXAMPLES: of other antibiotics from this class are Oxycycline, Doxycycline It is bacteriostatic antibiotic: reversibly binds to 30S ribosomal subunit ,inhibits tRNA binding to ribosome It is broad-spectrum antibiotic that is effective against Gram-positive and Gram-negative bacteria, not effective against Acid-fast bacteria. Side effects: Tetracyclines can cause discoloration in teeth when it is used by young children Bacterial resistance: Microbes reduce drug’s uptake or increase the antibiotic excretion. Erythromycin Class – macrolides; EXAMPLES: of other antibiotics from this class are Azithromycin, Lincomycin It is bacteriostatic antibiotic as it reversibly binds to 50S ribosomal subunit and inhibits translocation of ribosome, thus inhibiting the elongation of polypeptide chain. It is narrow-spectrum antibiotic that is effective against Gram-positive and Gram-negative bacteria. Bacterial resistance: Microbe produce enzyme that chemically modifies 23S ribosomal RNA via methylation. Chloramphenicol Class – chloramphenicols; It is bacteriostatic antibiotic that inhibits bacterial growth by reversible binding to 50S ribosomal subunit and blocking peptide bond formation, thus inhibiting the elongation of polypeptide chain. It is broad-spectrum antibiotic that effective against Gram-positive and Gram-negative bacteria. Side effects: It is toxic and reserved as the last resort antibiotic. It can cause fatal aplastic anemia, a condition when bone marrow does not produce enough new blood cells of all types to replenish the blood. It also increases the risk of leukemia. Bacterial resistance: Microbe produce enzyme that acetylates the antibiotic. Andrei V. Nikonov, Ph.D. 25 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Inhibition of nucleic acid synthesis Disruption of cell membrane Fluoroquinolones integrity ‒ Ciprofloxacin is o Irreversibly binding to DNA gyrase These medications are causing the cells to  Inhibits DNA synthesis by preventing topoisomerases leak, leading to cell death. from relieving the strain that appears during replication ‒ Due to similarity in structure of membranes o Bactericidal of prokaryotic and eukaryotic cells, the use o Broad-spectrum of these medications is primarily limited to  Effective against any bacteria, Gram-positive, Gram- apical applications. negative, and Acid-fast ‒ Daptomycin is o Side effects: various, including cardiac arrhythmia o Bactericidal o Narrow-spectrum Rifamycins  Effective against Gram-positive ‒ Rifampin is bacteria o Irreversibly binding to RNA polymerase  Inhibits RNA synthesis by preventing RNA polymerase ‒ Polymyxin is from initiation of transcription o Bactericidal o Bactericidal o Narrow-spectrum o Broad-spectrum  Effective against Gram-negative  Effective against any bacteria, Gram-positive, Gram- bacteria negative, acid-fast o Side effects: hepatitis, flu-like symptoms 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 26 Ciprofloxacin Class – fluoroquinolones. EXAMPLES: of other antibiotics from this class are Gemifloxacin, Levifloxacin It is bactericidal antibiotic as it irreversibly binds to DNA gyrase and other enzymes of DNA synthesis. It is broad spectrum antibiotic that effective against Gram-positive, Gram-negative and Acid-fast bacteria Side effects: due to serious side effects, including increased risk of cancer, its medical use is limited to treatment of tuberculosis. Bacterial resistance: Genetic mutations leading to changes in targeted enzymes. Microbial resistance can develop rapidly, even during a course of treatment. Rifampin (Rifampicin) Class – rifamycins. EXAMPLES: of other antibiotics from this class are Rifabutin, Rifaximin It is bactericidal antibiotic as it irreversibly binds to RNA polymerase and inhibits RNA synthesis. It is broad spectrum antibiotic that effective against Gram-positive, Gram-negative and Acid-fast bacteria. Side effects: Due to high toxicity, it’s medical use is limited to treatment of tuberculosis, leprosy and prophylaxis of meningitis in people exposed to Neisseria meningitidis. Bacterial resistance: Genetic mutations leading to changes in targeted enzymes. Andrei V. Nikonov, Ph.D. 26 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Inhibition of metabolic pathways PABA (precursor #1) Sulfonamides ‒ Sulfa drugs are Enzyme # 1 Sulfa drugs o Competitive inhibitors of dihydropteroate synthetase  Inhibit folic acid biosynthesis by competing with PABA for active site of the enzyme Precursor # 2, PABA is a o Bacteriostatic substrate for o Broad-spectrum Enzyme # 2 dihydropteroate  Effective against any bacteria, Gram-positive or Gram-negative synthase o Side effects: toxic epidermal necrosis, hemolytic (enzyme # 1) Dihyrdofolate anemia, Steven-Johnson syndrome Trimethoprim Enzyme # 3 Trimethoprim ‒ Trimethoprim is o Competitive inhibitor of dihydrofolate reductase Tetrahyrdofolate  Inhibit folic acid biosynthesis by reversible binding to active site of the enzyme o Bacteriostatic Multiple enzymes o Broad-spectrum and reactions  Effective against any bacteria, Gram-positive or Sulfanilamide Gram-negative T, G, A nucleotides Coenzymes is a competitive o Side effects: diarrhea, nausea, sun sensitivity inhibitor 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 27 Folic acid It is precursor of nucleotides and also used in synthesis of coenzymes Folic acid biosynthesis pathway is present in bacterial cells. Folic acid biosynthesis pathway is not present in humans. As a result, humans have to get folic acid from the food Sulfanilamide It is competitive inhibitor of dihydropteroate synthetase, the enzyme catalyzing the first step of metabolic pathway of folic acid biosynthesis Substrate of dihydropteroate synthetase is PABA Sulfanilamide has structural similarities to PABA It competes with PABA for binding to active site of dihydropteroate synthetase. Sulfanilamide is effective antimicrobial drug. Antibiotic has very high therapeutic index Combined use of sulfanilamide and trimethoprim has synergistic effect Trimethoprim inhibits another enzyme of metabolic pathway of folic acid biosynthesis Mechanism of microbial resistance: Acquisition of the plasmid carrying the genes of alternative enzymes Side effects relatively rare: Stevens Johnson syndrome – necrosis of epidermal cells leading to separation of dermis from epidermis. Type I allergy Q. Risks of microbial resistance development are dramatically reduced if sulfanilamide and trimethoprim are used together. Can you explain why? Andrei V. Nikonov, Ph.D. 27 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 Acting against Mycobacterium tuberculosis Bacterial species from genus Mycobacterium have acid-fast type of bacterial cell wall ‒ The wall is highly hydrophobic due to presence of mycolic acids ‒ It is extremely hard for antibacterial medications penetrate the wall To treat the infections caused by Mycobacterium spp., a special strategy is used along with cocktail of bactericidal antibiotics: ‒ Antibiotics that reduce hydrophobicity of acid-fast cell wall: o Ethambutol prevents the attachment of mycolic acids to cell wall  Bacteriostatic o Isoniazid inhibits the synthesis of mycolic acid  Bactericidal o Pyrazinamide probably inhibits the fatty acid synthesis  Bactericidal ‒ Bactericidal antibiotics that kill the bacteria: o Streptomycin irreversibly binds to ribosome & inhibits protein synthesis – Bactericidal o Rifampin irreversibly binds to RNA polymerase & inhibits RNA synthesis – Bactericidal o Ciprofloxacin irreversibly binds to DNA gyrase & inhibits DNA synthesis – Bactericidal ‒ Directly Observed Therapy (DOT) o See slide notes for details 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 28 Ethambutol It is bacteriostatic against actively growing Mycobacterium tuberculosis. It reduces hydrophobicity of cell wall by inhibiting the enzyme arabinosyl transferase involved in synthesis of arabinogalactan and thus preventing the attachment of mycolic acids to the cell wall. It has various side effects including Optic neuritis; Red-green blindness; Peripheral neuropathy; Damage to liver; Joint pain Isoniazid It is bactericidal to rapidly growing mycobacteria but bacteriostatic if mycobacteria are slow-growing. It is prodrug, which is activated in the bacterial cell It inhibits the mycolic acid synthesis Pyrazinamide It is bactericidal to rapidly growing mycobacteria but bacteriostatic if mycobacteria are slow-growing. It is a prodrug, which is activated in the bacterial cell Mechanism of action is not clear but may be involve inhibition of fatty acid synthase and thus interfering with synthesis of long chain (C16) fatty acids. DOT It means that a trained health care worker or other designated individual (excluding a family member) provides the prescribed TB drugs and watches the patient swallows every dose. Strategy Mycobacteria are acid-fast bacteria with highly hydrophobic cell wall. So, mycobacterial infections must be treated by cocktail of antibiotics for a few months and up to 1-2 years. The cocktail must include antibiotics that reduce hydrophobicity of cell wall and bactericidal antibiotics that can kill bacterial cells even at low concentration. Effective concentration of bacteriostatic antibiotics is impossible to achieve in acid-fast bacteria. At the beginning of treatment, antibiotics must be taken in the presence of healthcare worker (directly observed therapy or DOT). Andrei V. Nikonov, Ph.D. 28 BMCC/CUNY -- Bio 420 (Microbiology) -- Lecture 08 7/26/2024 20.4. Antimicrobial susceptibility testing Susceptibility to a given antimicrobial drug varies greatly between microbial species ‒ Therefore, before clinical use each drug must be tested in laboratory settings, followed by clinical trials Disk diffusion test MIC and MBC Effects of combination Paper disks containing drugs being The same amount of microbe is Mixing the antimicrobial drugs may tested laid over bacterial lawn and added to set of test-tube with serially affect their performance incubated diluted drug ‒ Antagonistic effect occurs when the ‒ Appearance of zones of inhibition ‒ Minimal Inhibitory Concentration outcome of mixed drugs is worse indicates antimicrobial activity of the (MIC) is lowest concentration of drug than the one of individual drug. drug at which microbe is not growing ‒ Synergistic effect occurs when the ‒ The bigger zone of inhibition, the Aliquots are taken from t-tube and outcome of mixed drugs is better than lower concentration of drug has plated on Petri dish w/o drug the one of individual drug. antimicrobial effect Minimal Bactericidal Concentration A B C Chloramphenicol (MBC) is lowest drug concentration at which all (99.9%) bacterial cells are killed Tetracycline Penicillin [high] [low] MIC Streptomycin What effect would you expect from E. coli B. subtilis combined sulfanilamide and trimethoprim? MBC 7/26/2024 Animated BMCC / CUNY -- Bio 420 (Microbiology) -- Lecture 08 See slide notes 29 Spectrum of activity of antimicrobial medications Broad and narrow spectrum drugs – for details, see slide notes to slide 14 It is checked by Kirby-Bauer (disk-diffusion) method For details, see slide 25 of lecture 2 The bigger zone of inhibition, the more effective drug is. However, size of inhibition zone is also influenced by the drug’s size, stability and concen