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MICROBIOLOGY FOR
          MEDICAL GRADUATES
                WHAT YOU SHOULD KNOW

                    Dr.T.V.Rao MD




DR.T.V.RAO MD                       11/16/2012   1
AIMS FOR LEARNING MEDICAL
               MICROBIOLOGY
• What is medical microbiology?
• Why is it relevant?
• Some important concepts.
• Basic classification of organisms.
• Classifying bacteria.

DR.T.V.RAO MD                    11/16/2012   2
WHAT IS MEDICAL MICROBIOLOGY?
      The study of microorganisms
        “


    (including bacteria, viruses, fungi
        and parasites) which are of
       medical importance and are
      capable of causing diseases in
             human beings”
DR.T.V.RAO MD                 11/16/2012   3
THE EARLY YEARS OF MICROBIOLOGY
    CONTRIBUTED BY DISCOVERY OF MICROSCOPE




DR.T.V.RAO MD                    11/16/2012   4
THE FIRST OBSERVATIONS
• 1673-1723, Antoni
  van Leeuwenhoek
  described live
  microorganisms that
  he observed in
  teeth scrapings, rain
  water, and
  peppercorn
  infusions.

  DR.T.V.RAO MD           11/16/2012       5
                                       Figure 1.2b
THE EARLY YEARS OF
                   MICROBIOLOGY
• How Can Microbes Be Classified?
     • Carolus Linnaeus (Swedish) developed taxonomic system for
       naming plants and animals and grouping similar organisms
       together
     • Leeuwenhoek’s microorganisms grouped into six categories as
       follows:
            • Fungi
            • Protozoa
            • Algae
            • Bacteria
            • Archaea
            • Small animals
 DR.T.V.RAO MD                                    11/16/2012       6
WHAT IS MEDICAL MICROBIOLOGY?
       THE PURPOSE OF LEARNING
What organisms cause
 infection?
How they cause infection.
How to treat them.
How to prevent infection.
DR.T.V.RAO MD            11/16/2012   7
WHY IS IT IMPORTANT?
• Infection is one of the most important
  causes of mortality and morbidity in the
  population.
• Approximately 30% of hospital patients
  are on antibiotics at any one time
• 1 in 10 patients acquires an infection
  whilst in hospital.
DR.T.V.RAO MD                   11/16/2012   8
THE HISTORICAL CONTRIBUTION IN THE
  SUBJECT OF MICROBIOLOGY BY …
                        Darwin
•   Linnaeus        •

                    •   Salk
•   Jenner          •   Watson & Crick
                    •   Jacob and Monod
•   Hooke           •   McClintock
                        Woese
•   Leeuwenhoek     •
                    •   Venter?
•   Lister
•   Pasteur
•   Koch

    DR.T.V.RAO MD                  11/16/2012   9
DEFINITIONS
• Bacteriology is the study of bacteria.
• Mycology is the study of fungi.
• Parasitology is the study of protozoa and parasitic worms.
• Recent advances in genomics, the study of an organism’s
  genes, have provided new tools for classifying
  microorganisms.
• Proteomics is looking at the gene products
    DR.T.V.RAO MD                          11/16/2012   10
LEARN THE CLASSIFICATION OF
             ORGANISMS
• All living organisms are classified into:
          • Kingdom
          • Phylum (family)
          • Genus
          • Species
• Organisms that can cause disease are many and varied and include:
          • Viruses
          • Bacteria
          • Fungi
          • Parasites
    DR.T.V.RAO MD                                  11/16/2012     11
RELEVANCE OF CLASSIFICATION
• Different:
      • Diseases
      • Modes of
        transmission
      • Treatment-e.g.
        routinely use
        antibiotics don’t cure
        vira lfungalinfections

DR.T.V.RAO MD                    11/16/2012   12
THE GOLDEN AGE OF MICROBIOLOGY
         LOUIS PASTEUR CHANGES THE FUTURE OF
                    MICROBIOLOGY




DR.T.V.RAO MD                        11/16/2012   13
FERMENTATION AND PASTEURIZATION
• Pasteur demonstrated that
  these spoilage bacteria could
  be killed by heat that was not
  hot enough to evaporate the
  alcohol in wine.
• Pasteurization is the
  application of a high heat for a
  short time.


  DR.T.V.RAO MD                      11/16/2012              14
                                                  Figure 1.4 (1 of 3)
THE GOLDEN AGE OF MICROBIOLOGY




DR.T.V.RAO MD             11/16/2012   15
THE GOLDEN AGE OF MICROBIOLOGY
• Koch’s Postulates

    • Suspected causative agent must be found in
      every case of the disease and be absent from
      healthy hosts
    • Agent must be isolated and grown outside the
      host
    • When agent is introduced into a healthy,
      susceptible host, the host must get the disease
    • Same agent must be reisolated from now-
      diseased experimental host
DR.T.V.RAO MD                           11/16/2012   16
NORMAL MICROBIOTA
• Normal Microbiota prevent growth of
  pathogens.
•   Normal Microbiota produce growth factors
    such as folic acid and vitamin K.
• Resistance is the ability of the body to
  ward off disease.
• Resistance factors include skin,
  stomach acid, and antimicrobial
  chemicals.
• Biofilms are extremely important in
  microbial ecology




     DR.T.V.RAO MD                             11/16/2012   17
NORMAL MICRO BIOTA ON THE HUMAN BODY




DR.T.V.RAO MD              11/16/2012
                                        Table 18
                                              14.1
NORMAL MICROBIOTA
• Animals, including humans, are usually germfree
  in utero.
• Microorganisms begin colonization in and on the
  surface of the body soon after birth.



• Microorganisms that establish permanent colonies
  inside or on the body without producing disease
  make up the normal microbiota.
• Transient microbiota are microbes that are
  present for various periods and then
  disappear.
  DR.T.V.RAO MD                     11/16/2012   19
WE HAVE MORE MICROBES OCCUPYING OUR BODY
           THAN OUR OWN CELLS




  DR.T.V.RAO MD              11/16/2012   20
CLASSIFYING BACTERIA
Why bother?
Different bacteria:
• cause different diseases
• are susceptible/resistant to
  different antibiotics
• some bacteria are common
  normal flora whilst other
  closely related species are
  pathogens


DR.T.V.RAO MD                    11/16/2012   21
CLASSIFYING BACTERIA
How?
• 1st into broad groups based
  on microscopic appearance
• Then divided into species
  based on a range of
  different properties-often
  biochemical reactions e.g.
  some may be able to
  metabolise a sugar that
  others cannot.


DR.T.V.RAO MD                   11/16/2012   22
GRAM STAIN
Method of differentiating bacteria.
Can be either Gram +ve or Gram –
   ve depending on how they
   appear with the stain.
Can then be further grouped based
   on shape (rod=long thin or
   coccus=round).
Thus we end up with 4
   combinations:
G+ rod, G+ coccus, G- rod, G-
   coccus



DR.T.V.RAO MD                         11/16/2012   23
BACTERIAL CELL WALL MAKES THE
            BASIC DIFFERENCE




DR.T.V.RAO MD               11/16/2012   24
GRAM STAIN
                                G+ve        G-ve
• STAIN the slide with
  crystal violet for 1-2 min.
• Flood slide with Gram's
  iodine for 1-2 min.
• Decolourise by washing
  the slide briefly with
  acetone (2-3 seconds).
• Stain with safranin
  counterstain for 2 min.
• View under microscope

 DR.T.V.RAO MD                         11/16/2012   25
GRAM STAIN
Gives an initial idea of the
   possible identity of the
   organism.
Can be done without growing
  the organism (i.e. rapid
  result)
Thus can be done on pus, joint
  fluid, sputum, CSF
1st result available on blood
    cultures

DR.T.V.RAO MD                    11/16/2012   26
GRAM STAIN
Relevance of Gram reaction.
• Gram +ve and gram –ve
  organisms ae susceptible
  to different groups of
  antibiotics.
• Cause different diseases
• Differ in their ability to
  survive in the environment-
  cleaning, infection control,
  outbreak management.

DR.T.V.RAO MD                    11/16/2012   27
GRAM POSITIVE COCCI
                                 • Clusters: usually
                                    characteristic of
                              Staphylococcus spp., such as
                                       S. aureus


                          •   Chain or pairs: usually characteristic
                                of Streptococcus spp., such as
                                         S. pneumoniae




DR.T.V.RAO MD                                11/16/2012          28
GRAM POSITIVE BACILLI
                • Thick : usually
                  characteristic of
                  Clostridium spp., such
                  as C. perfringens, C.
                  difficle, C. tetani




                • Thin: e.g. Listeria spp.


DR.T.V.RAO MD               11/16/2012     29
GRAM NEGATIVE BACILLI
                           • Thin rods: usually
                             characteristic of
                             enterobacteriaceae
                             (coliforms), such as E. Coli



                           • Coccobacilli: usually
                             characteristic of
                             Haemophilus spp., such as
                             H. influenzae


DR.T.V.RAO MD                            11/16/2012     30
GRAM NEGATIVE BACILLI
                • Curved: usually
                  characteristic of Vibrio
                  spp.or Campylobacter spp.,
                  such as V. cholerae C. jejuni



                • Thin needle shape: usually
                  characteristic of
                  Fusobacterium spp.



DR.T.V.RAO MD                 11/16/2012     31
GRAM NEGATIVE COCCI
                          • Diplococci: usually characteristic of
                            Neisseria spp., such as N.
                            meningitides or N. gonorrhea.
                            Though In addition, Moraxella spp.
                            and Acinetobacter spp.are often
                            diplococcal in morphology.



                          • Coccobacilli: usually characteristic
                            of Acinetobacter spp., which can be
                            either Gram-positive or Gram-
                            negative, and is often called Gram-
                            variable.



DR.T.V.RAO MD                                11/16/2012        32
WHAT CAN YOU SEE ON THE SLIDE?
1.    Gram +ve cocci
2.    Gram +ve bacilli
3.    Gram –ve cocci
4.    Gram –ve bacilli


                                              53%
                  47%




                                   0%                   0%


                                                              Staphylococcus aureus – 100x
                  i



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                                ci




                                                       ci
             co




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                                        –v
                         +v




                                                  –v
         m




                                       m
                         m




                                                 m
      ra




                                    ra
                      ra




                                              ra
      G




                                  G
                  G




                                              G




 DR.T.V.RAO MD                                                               11/16/2012      33
WHAT CAN YOU SEE ON THE SLIDE?
1.          Gram +ve cocci
2.          Gram +ve bacilli                                 Streptococcus pneumoniae
3.          Gram –ve cocci
4.          Gram –ve bacilli
                                 68%




                 16%
                                                       11%
                                             5%
                 i



                                 lli




                                                       lli
                                             i
                cc




                                            cc
                               ci




                                                      ci
            co




                                          co
                            ba




                                                    ba
            e




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                                                   e
         +v




                                       –v
                        +v




                                                 –v
        m




                                      m
                        m




                                                m
     ra




                                   ra
                     ra




                                             ra
     G




                                 G
                 G




                                             G




 DR.T.V.RAO MD                                                         11/16/2012   34
WHAT CAN YOU SEE ON THE SLIDE?
1.       Gram +ve cocci
2.       Gram +ve bacilli
3.       Gram –ve cocci
4.       Gram –ve bacilli

                                             36% 36%




                 14% 14%


                                                             Pseudomonas aeruginosa
                 i



                                 lli




                                                       lli
                                             i
                cc




                                            cc
                               ci




                                                      ci
            co




                                          co
                            ba




                                                    ba
            e




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                           e




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         +v




                                       –v
                        +v




                                                 –v
        m




                                      m
                        m




                                                m
     ra




                                   ra
                     ra




                                             ra
     G




                                 G
                 G




                                             G




 DR.T.V.RAO MD                                                         11/16/2012     35
VIRUSES
Small (50-300nm)
Unable to replicate
independently
Invade host cells and use
their cellular machinery to
replicate
Influenza, Chickenpox
(varicella), Herpes,
Rhinovirus, HIV/AIDS
Often difficult to treat
  DR.T.V.RAO MD                 11/16/2012   36
FUNGI
•     Complex, large organisms
•     Eukaryotes (as are humans!)
•     Divided into yeasts & moulds
•     Cause a range of diseases
      e.g.:
        • Thrush
        • Athletes foot
        • Invasive & allergic aspergillosis
• Many diseases are
  opportunistic.



    DR.T.V.RAO MD                             11/16/2012   37
PROTOZOA

• Eukaryotes
• Absorb or ingest
  organic chemicals
• May be motile via
  pseudopods, cilia,
  or flagella

  DR.T.V.RAO MD              11/16/2012      38
                                          Figure 1.1c
MULTICELLULAR ANIMAL
                      PARASITES
                                   •   Eukaryote
                              •   Multicellular animals
            •   Parasitic flatworms and round worms are called Helminths.
                        •   Microscopic stages in life cycles.




DR.T.V.RAO MD                                                      11/16/2012        39
                                                                                Figure 12.28a
THE ETIOLOGY OF INFECTIOUS DISEASES


•     Koch’s postulates are criteria for establishing that
      specific microbes cause specific diseases.
•     Koch’s postulates have the following requirements:
(a) the same pathogen must be present in every case of
    the disease;
(b) the pathogen must be isolated in pure culture;
(c) the pathogen isolated from pure culture must cause the
    same disease in a healthy, susceptible laboratory
    animal;
(d) the pathogen must be reisolated from the inoculated
    laboratory animal.

    DR.T.V.RAO MD                                11/16/2012   40
KOCH’S POSTULATES




DR.T.V.RAO MD       11/16/2012           41
                                 Figure 14.7
EXCEPTIONS TO KOCH’S POSTULATES
• Koch’s postulates are modified to establish etiologies of
  diseases caused by viruses and some bacteria, which cannot
  be grown on artificial media.
• Some diseases, such as tetanus, have unequivocal signs
  and symptoms.
• Some diseases, such as pneumonia and nephritis, may be
  caused by a variety of microbes.
• Some pathogens, such as S. pyrogenes, cause several
  different diseases.
• Certain pathogens, such as HIV, cause disease in humans
  only.

  DR.T.V.RAO MD                            11/16/2012   42
Diseases and Infections
 • Disease-causing microorganisms are
   called pathogens.
 • Pathogenic microorganisms have special
   properties that allow them to invade the
   human body or produce toxins.
 • When a microorganism overcomes the
   body’s defenses, a state of disease
   results.
DR.T.V.RAO MD                    11/16/2012   43
PATHOLOGY, INFECTION, AND DISEASE
• Pathology is the scientific study of disease.
• Pathology is concerned with the
    • etiology (cause),
    • pathogenesis (development),
    • effects of disease – structural and functional changes brought
      about by disease.
• Infection is the invasion and growth of pathogens in the body.
• A host is an organism that shelters and supports the growth of
  pathogens.
• Disease is an abnormal state in which part or all of the body is not
  properly adjusted or is incapable of performing normal functions.
• Infection disease – presence of particular microorganism in part of
  the body where is not usually found.
   DR.T.V.RAO MD                                    11/16/2012      44
IMMUNITY PROTECTS FROM EVENTS WITH
               INFECTIONS




DR.T.V.RAO MD                11/16/2012   45
CLASSIFYING INFECTIOUS DISEASES

• Every disease alters body structures and functions
• A patient may exhibit
   • symptoms (subjective changes in body functions)
          • Pain or body discomfort
   • signs (measurable changes), which a physician uses to
     make a diagnosis (identification of the disease)
          • Fever, swelling, paralysis

• A specific group of symptoms or signs that always
  accompanies a specific disease is called a
  syndrome.


  DR.T.V.RAO MD                           11/16/2012   46
MICROORGANISMS




DR.T.V.RAO MD                11/16/2012     47
                                          Figure 1.1
CLASSIFYING INFECTIOUS DISEASES
• Communicable diseases are transmitted directly
  or indirectly from one host to another.
                 • Chicken pox, genital herpes,
   • A contagious disease is one that is easily
     spread from one person to another.
• Noncommunicable diseases are caused by
  microorganisms that normally grow outside the
  human body and are not transmitted from one
  host to another
          • Tetanus, Clostridium tetani

 DR.T.V.RAO MD                                11/16/2012   48
THE MODERN AGE OF MICROBIOLOGY




DR.T.V.RAO MD             11/16/2012   49
THE MODERN AGE OF MICROBIOLOGY
• Microbial Genetics
     • Avery, MacLeod, and McCarty determined genes are
       contained in molecules of DNA
     • Beadle and Tatum established that a gene’s activity is
       related to protein function
     • Translation of genetic information into protein
       explained
     • Rates and mechanisms of genetic mutation
       investigated
     • Control of genetic expression by cells described

 DR.T.V.RAO MD                                  11/16/2012   50
THE MODERN AGE OF MICROBIOLOGY
• Molecular Biology
     • Explanation of cell function at the molecular level
     • Genome sequencing
     • Pauling proposed that gene sequences could
            • Provide understanding of evolutionary relationships and processes
            • Establish taxonomic categories that reflect these relationships
            • Identify existence of microbes that have never been cultured
     • Woese determined that cells belong to bacteria, archaea, or eukaryotes
     • Cat-scratch fever caused by unculturable organism




 DR.T.V.RAO MD                                                   11/16/2012       51
THE MODERN AGE OF
                      MICROBIOLOGY
   •     Recombinant DNA Technology
       • Genes in microbes, plants, and
         animals manipulated for practical
         applications
       • Production of human blood-
         clotting factor by E. coli to aid
         hemophiliacs
                •   Gene Therapy
       • Inserting a missing gene or
         repairing a defective one in
         humans by inserting desired gene
         into host cells




DR.T.V.RAO MD                                11/16/2012   52
DISCOVERY OF ANTIMICROBIAL AGENTS
                                                  _________
  Alexander Fleming (1881 – 1955), a
   Scottish biologist and
   pharmacologist, observed
   bacterial staphylococci colonies
   disappearing on plates
   contaminated with mold.
  Fleming extracted the
   compound from the mold
   responsible for destruction of
   the bacterial colonies.
  The product of the mold was
   named penicillin, after the
   Penicillium mold from which it
   was derived.
  Nobel Prize in Physiology of
   Medicine in 1945.

                                                                   Images: Penicillium mold, PHIL #8396; Staphylococcus aureus on
         DR.T.V.RAO MD                                                                       11/16/2012                  53
                                                                    antibiotic test plate, PHIL #2641; Poster attached to a mailbox
From the Virtual Microbiology Classroom on ScienceProfOnline.com            offering advice to World War II servicemen, 1944, NIH
THE MODERN AGE OF
                     MICROBIOLOGY
• How Do We Defend Against Disease?
     • Serology
            • The study of blood serum
            • Von Behring and Kitasato – existence in the blood of
              chemicals and cells that fight infection
     • Immunology
            • The study of the body’s defense against specific pathogens
     • Chemotherapy
            • Fleming discovered penicillin
            • Domagk discovered sulfa drugs
 DR.T.V.RAO MD                                          11/16/2012    54
THE BIRTH OF MODERN
                    CHEMOTHERAPY
• Treatment with chemicals is chemotherapy.
• Chemotherapeutic agents used to treat infectious disease
  can be synthetic drugs or antibiotics.
• Antibiotics are chemicals produced by bacteria and fungi
  that inhibit or kill other microbes.
• Quinine from tree bark was long used to treat malaria.
• 1910: Paul Ehrlich developed a synthetic arsenic drug,
  salvarsan, to treat syphilis.
• 1930s: Sulfonamides were synthesized.
  DR.T.V.RAO MD                               11/16/2012     55
THE BIRTH OF MODERN CHEMOTHERAPY
 • 1928: Alexander
   Fleming discovered the
   first antibiotic.
 • He observed that
   Penicillium fungus
   made an antibiotic,
   penicillin, that killed S.
   aureus.
 • 1940s: Penicillin was
   tested clinically and
   mass produced.
 DR.T.V.RAO MD                  11/16/2012   56
MODERN DEVELOPMENTS IN MICROBIOLOGY
• Immunology is the study of
  immunity. Vaccines and
  interferons are being investigated
  to prevent and cure viral
  diseases.
• The use of immunology to identify
  some bacteria according to
  serotypes (variants within a
  species) was proposed by
  Rebecca Lancefield in 1933.
  DR.T.V.RAO MD                        11/16/2012              57
                                                    Figure 1.4 (3 of 3)
MODERN BIOTECHNOLOGY AND GENETIC
               ENGINEERING
• Biotechnology, the use of
  microbes to produce foods
  and chemicals, is centuries
  old.
• Genetic engineering is a
  new technique for
  biotechnology. Through
  genetic engineering,
  bacteria and fungi can
  produce a variety of
  proteins including vaccines
  and enzymes.
DR.T.V.RAO MD                   11/16/2012   58
SELECTED NOBEL PRIZES IN PHYSIOLOGY
                    OR MEDICINE
    1901*         von Behring                Diphtheria antitoxin
    1902          Ross                       Malaria transmission
    1905          Koch                       TB bacterium
    1908          Metchnikoff                Phagocytes
    1945          Fleming, Chain, Florey     Penicillin
    1952          Waksman                    Streptomycin
    1969          Delbrück, Hershey, Luria   Viral replication
    1987          Tonegawa                   Antibody genetics
.
    1997          Prusiner                   Prions




      DR.T.V.RAO MD                               11/16/2012   59
SELECTED NOVEL PRIZES IN PHYSIOLOGY OR
                   MEDICINE
1901*     von Behring       Diphtheria antitoxin
1902      Ross              Malaria transmission
1905      Koch                                  TB bacterium
1908      Metchnikoff                           Phagocytes
1945      Fleming, Chain, Florey                Penicillin
1952      Waksman                               Streptomycin
1969      Delbrück, Hershey, Luria              Viral replication
1987      Tonegawa                              Antibody genetics
1997Prusiner                                    Prions
2003Agre, Mackirron                   water and ion channels
2005 Marshall, Warren                  Helicobacter and ulcers
2008 Hausen                             Papilloma and viruses
  * The first Nobel Prize in Physiology or Medicine.
      DR.T.V.RAO MD                                      11/16/2012   60
UNIVERSAL PRECAUTIONS SET
             UP BY CDC
• Use gloves, gowns, masks and goggles
• Minimize risk of needle sticks
• Disinfections procedure
• Preventative treatment after exposure
• Reduce risk
• Treat all patients the same
• HBV greater risk than HIV
  DR.T.V.RAO MD                           11/16/2012   61
DEAR STUDENTS NEVER FORGET TO WASH HANDS
   AFTER HANDLING PATIENTS OR INFECTED
                MATERIAL




DR.T.V.RAO MD                 11/16/2012   62
VISIT ME FOR MORE ARTICLES OF INTEREST ON
      MICROBIOLOGY, INFECTIOUS DISEASES…




DR.T.V.RAO MD                     11/16/2012   63
• Programme Created by Dr.T.V.Rao MD
        for Undergraduate Medical and
    Paramedical Students for orientation in
        Learning Medical Microbiology
                        • email
                • doctortvrao@gmail.com


DR.T.V.RAO MD                        11/16/2012   64

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Microbiology for medical graduates

  • 1. MICROBIOLOGY FOR MEDICAL GRADUATES WHAT YOU SHOULD KNOW Dr.T.V.Rao MD DR.T.V.RAO MD 11/16/2012 1
  • 2. AIMS FOR LEARNING MEDICAL MICROBIOLOGY • What is medical microbiology? • Why is it relevant? • Some important concepts. • Basic classification of organisms. • Classifying bacteria. DR.T.V.RAO MD 11/16/2012 2
  • 3. WHAT IS MEDICAL MICROBIOLOGY? The study of microorganisms “ (including bacteria, viruses, fungi and parasites) which are of medical importance and are capable of causing diseases in human beings” DR.T.V.RAO MD 11/16/2012 3
  • 4. THE EARLY YEARS OF MICROBIOLOGY CONTRIBUTED BY DISCOVERY OF MICROSCOPE DR.T.V.RAO MD 11/16/2012 4
  • 5. THE FIRST OBSERVATIONS • 1673-1723, Antoni van Leeuwenhoek described live microorganisms that he observed in teeth scrapings, rain water, and peppercorn infusions. DR.T.V.RAO MD 11/16/2012 5 Figure 1.2b
  • 6. THE EARLY YEARS OF MICROBIOLOGY • How Can Microbes Be Classified? • Carolus Linnaeus (Swedish) developed taxonomic system for naming plants and animals and grouping similar organisms together • Leeuwenhoek’s microorganisms grouped into six categories as follows: • Fungi • Protozoa • Algae • Bacteria • Archaea • Small animals DR.T.V.RAO MD 11/16/2012 6
  • 7. WHAT IS MEDICAL MICROBIOLOGY? THE PURPOSE OF LEARNING What organisms cause infection? How they cause infection. How to treat them. How to prevent infection. DR.T.V.RAO MD 11/16/2012 7
  • 8. WHY IS IT IMPORTANT? • Infection is one of the most important causes of mortality and morbidity in the population. • Approximately 30% of hospital patients are on antibiotics at any one time • 1 in 10 patients acquires an infection whilst in hospital. DR.T.V.RAO MD 11/16/2012 8
  • 9. THE HISTORICAL CONTRIBUTION IN THE SUBJECT OF MICROBIOLOGY BY … Darwin • Linnaeus • • Salk • Jenner • Watson & Crick • Jacob and Monod • Hooke • McClintock Woese • Leeuwenhoek • • Venter? • Lister • Pasteur • Koch DR.T.V.RAO MD 11/16/2012 9
  • 10. DEFINITIONS • Bacteriology is the study of bacteria. • Mycology is the study of fungi. • Parasitology is the study of protozoa and parasitic worms. • Recent advances in genomics, the study of an organism’s genes, have provided new tools for classifying microorganisms. • Proteomics is looking at the gene products DR.T.V.RAO MD 11/16/2012 10
  • 11. LEARN THE CLASSIFICATION OF ORGANISMS • All living organisms are classified into: • Kingdom • Phylum (family) • Genus • Species • Organisms that can cause disease are many and varied and include: • Viruses • Bacteria • Fungi • Parasites DR.T.V.RAO MD 11/16/2012 11
  • 12. RELEVANCE OF CLASSIFICATION • Different: • Diseases • Modes of transmission • Treatment-e.g. routinely use antibiotics don’t cure vira lfungalinfections DR.T.V.RAO MD 11/16/2012 12
  • 13. THE GOLDEN AGE OF MICROBIOLOGY LOUIS PASTEUR CHANGES THE FUTURE OF MICROBIOLOGY DR.T.V.RAO MD 11/16/2012 13
  • 14. FERMENTATION AND PASTEURIZATION • Pasteur demonstrated that these spoilage bacteria could be killed by heat that was not hot enough to evaporate the alcohol in wine. • Pasteurization is the application of a high heat for a short time. DR.T.V.RAO MD 11/16/2012 14 Figure 1.4 (1 of 3)
  • 15. THE GOLDEN AGE OF MICROBIOLOGY DR.T.V.RAO MD 11/16/2012 15
  • 16. THE GOLDEN AGE OF MICROBIOLOGY • Koch’s Postulates • Suspected causative agent must be found in every case of the disease and be absent from healthy hosts • Agent must be isolated and grown outside the host • When agent is introduced into a healthy, susceptible host, the host must get the disease • Same agent must be reisolated from now- diseased experimental host DR.T.V.RAO MD 11/16/2012 16
  • 17. NORMAL MICROBIOTA • Normal Microbiota prevent growth of pathogens. • Normal Microbiota produce growth factors such as folic acid and vitamin K. • Resistance is the ability of the body to ward off disease. • Resistance factors include skin, stomach acid, and antimicrobial chemicals. • Biofilms are extremely important in microbial ecology DR.T.V.RAO MD 11/16/2012 17
  • 18. NORMAL MICRO BIOTA ON THE HUMAN BODY DR.T.V.RAO MD 11/16/2012 Table 18 14.1
  • 19. NORMAL MICROBIOTA • Animals, including humans, are usually germfree in utero. • Microorganisms begin colonization in and on the surface of the body soon after birth. • Microorganisms that establish permanent colonies inside or on the body without producing disease make up the normal microbiota. • Transient microbiota are microbes that are present for various periods and then disappear. DR.T.V.RAO MD 11/16/2012 19
  • 20. WE HAVE MORE MICROBES OCCUPYING OUR BODY THAN OUR OWN CELLS DR.T.V.RAO MD 11/16/2012 20
  • 21. CLASSIFYING BACTERIA Why bother? Different bacteria: • cause different diseases • are susceptible/resistant to different antibiotics • some bacteria are common normal flora whilst other closely related species are pathogens DR.T.V.RAO MD 11/16/2012 21
  • 22. CLASSIFYING BACTERIA How? • 1st into broad groups based on microscopic appearance • Then divided into species based on a range of different properties-often biochemical reactions e.g. some may be able to metabolise a sugar that others cannot. DR.T.V.RAO MD 11/16/2012 22
  • 23. GRAM STAIN Method of differentiating bacteria. Can be either Gram +ve or Gram – ve depending on how they appear with the stain. Can then be further grouped based on shape (rod=long thin or coccus=round). Thus we end up with 4 combinations: G+ rod, G+ coccus, G- rod, G- coccus DR.T.V.RAO MD 11/16/2012 23
  • 24. BACTERIAL CELL WALL MAKES THE BASIC DIFFERENCE DR.T.V.RAO MD 11/16/2012 24
  • 25. GRAM STAIN G+ve G-ve • STAIN the slide with crystal violet for 1-2 min. • Flood slide with Gram's iodine for 1-2 min. • Decolourise by washing the slide briefly with acetone (2-3 seconds). • Stain with safranin counterstain for 2 min. • View under microscope DR.T.V.RAO MD 11/16/2012 25
  • 26. GRAM STAIN Gives an initial idea of the possible identity of the organism. Can be done without growing the organism (i.e. rapid result) Thus can be done on pus, joint fluid, sputum, CSF 1st result available on blood cultures DR.T.V.RAO MD 11/16/2012 26
  • 27. GRAM STAIN Relevance of Gram reaction. • Gram +ve and gram –ve organisms ae susceptible to different groups of antibiotics. • Cause different diseases • Differ in their ability to survive in the environment- cleaning, infection control, outbreak management. DR.T.V.RAO MD 11/16/2012 27
  • 28. GRAM POSITIVE COCCI • Clusters: usually characteristic of Staphylococcus spp., such as S. aureus • Chain or pairs: usually characteristic of Streptococcus spp., such as S. pneumoniae DR.T.V.RAO MD 11/16/2012 28
  • 29. GRAM POSITIVE BACILLI • Thick : usually characteristic of Clostridium spp., such as C. perfringens, C. difficle, C. tetani • Thin: e.g. Listeria spp. DR.T.V.RAO MD 11/16/2012 29
  • 30. GRAM NEGATIVE BACILLI • Thin rods: usually characteristic of enterobacteriaceae (coliforms), such as E. Coli • Coccobacilli: usually characteristic of Haemophilus spp., such as H. influenzae DR.T.V.RAO MD 11/16/2012 30
  • 31. GRAM NEGATIVE BACILLI • Curved: usually characteristic of Vibrio spp.or Campylobacter spp., such as V. cholerae C. jejuni • Thin needle shape: usually characteristic of Fusobacterium spp. DR.T.V.RAO MD 11/16/2012 31
  • 32. GRAM NEGATIVE COCCI • Diplococci: usually characteristic of Neisseria spp., such as N. meningitides or N. gonorrhea. Though In addition, Moraxella spp. and Acinetobacter spp.are often diplococcal in morphology. • Coccobacilli: usually characteristic of Acinetobacter spp., which can be either Gram-positive or Gram- negative, and is often called Gram- variable. DR.T.V.RAO MD 11/16/2012 32
  • 33. WHAT CAN YOU SEE ON THE SLIDE? 1. Gram +ve cocci 2. Gram +ve bacilli 3. Gram –ve cocci 4. Gram –ve bacilli 53% 47% 0% 0% Staphylococcus aureus – 100x i lli lli i cc cc ci ci co co ba ba e e e e +v –v +v –v m m m m ra ra ra ra G G G G DR.T.V.RAO MD 11/16/2012 33
  • 34. WHAT CAN YOU SEE ON THE SLIDE? 1. Gram +ve cocci 2. Gram +ve bacilli Streptococcus pneumoniae 3. Gram –ve cocci 4. Gram –ve bacilli 68% 16% 11% 5% i lli lli i cc cc ci ci co co ba ba e e e e +v –v +v –v m m m m ra ra ra ra G G G G DR.T.V.RAO MD 11/16/2012 34
  • 35. WHAT CAN YOU SEE ON THE SLIDE? 1. Gram +ve cocci 2. Gram +ve bacilli 3. Gram –ve cocci 4. Gram –ve bacilli 36% 36% 14% 14% Pseudomonas aeruginosa i lli lli i cc cc ci ci co co ba ba e e e e +v –v +v –v m m m m ra ra ra ra G G G G DR.T.V.RAO MD 11/16/2012 35
  • 36. VIRUSES Small (50-300nm) Unable to replicate independently Invade host cells and use their cellular machinery to replicate Influenza, Chickenpox (varicella), Herpes, Rhinovirus, HIV/AIDS Often difficult to treat DR.T.V.RAO MD 11/16/2012 36
  • 37. FUNGI • Complex, large organisms • Eukaryotes (as are humans!) • Divided into yeasts & moulds • Cause a range of diseases e.g.: • Thrush • Athletes foot • Invasive & allergic aspergillosis • Many diseases are opportunistic. DR.T.V.RAO MD 11/16/2012 37
  • 38. PROTOZOA • Eukaryotes • Absorb or ingest organic chemicals • May be motile via pseudopods, cilia, or flagella DR.T.V.RAO MD 11/16/2012 38 Figure 1.1c
  • 39. MULTICELLULAR ANIMAL PARASITES • Eukaryote • Multicellular animals • Parasitic flatworms and round worms are called Helminths. • Microscopic stages in life cycles. DR.T.V.RAO MD 11/16/2012 39 Figure 12.28a
  • 40. THE ETIOLOGY OF INFECTIOUS DISEASES • Koch’s postulates are criteria for establishing that specific microbes cause specific diseases. • Koch’s postulates have the following requirements: (a) the same pathogen must be present in every case of the disease; (b) the pathogen must be isolated in pure culture; (c) the pathogen isolated from pure culture must cause the same disease in a healthy, susceptible laboratory animal; (d) the pathogen must be reisolated from the inoculated laboratory animal. DR.T.V.RAO MD 11/16/2012 40
  • 41. KOCH’S POSTULATES DR.T.V.RAO MD 11/16/2012 41 Figure 14.7
  • 42. EXCEPTIONS TO KOCH’S POSTULATES • Koch’s postulates are modified to establish etiologies of diseases caused by viruses and some bacteria, which cannot be grown on artificial media. • Some diseases, such as tetanus, have unequivocal signs and symptoms. • Some diseases, such as pneumonia and nephritis, may be caused by a variety of microbes. • Some pathogens, such as S. pyrogenes, cause several different diseases. • Certain pathogens, such as HIV, cause disease in humans only. DR.T.V.RAO MD 11/16/2012 42
  • 43. Diseases and Infections • Disease-causing microorganisms are called pathogens. • Pathogenic microorganisms have special properties that allow them to invade the human body or produce toxins. • When a microorganism overcomes the body’s defenses, a state of disease results. DR.T.V.RAO MD 11/16/2012 43
  • 44. PATHOLOGY, INFECTION, AND DISEASE • Pathology is the scientific study of disease. • Pathology is concerned with the • etiology (cause), • pathogenesis (development), • effects of disease – structural and functional changes brought about by disease. • Infection is the invasion and growth of pathogens in the body. • A host is an organism that shelters and supports the growth of pathogens. • Disease is an abnormal state in which part or all of the body is not properly adjusted or is incapable of performing normal functions. • Infection disease – presence of particular microorganism in part of the body where is not usually found. DR.T.V.RAO MD 11/16/2012 44
  • 45. IMMUNITY PROTECTS FROM EVENTS WITH INFECTIONS DR.T.V.RAO MD 11/16/2012 45
  • 46. CLASSIFYING INFECTIOUS DISEASES • Every disease alters body structures and functions • A patient may exhibit • symptoms (subjective changes in body functions) • Pain or body discomfort • signs (measurable changes), which a physician uses to make a diagnosis (identification of the disease) • Fever, swelling, paralysis • A specific group of symptoms or signs that always accompanies a specific disease is called a syndrome. DR.T.V.RAO MD 11/16/2012 46
  • 47. MICROORGANISMS DR.T.V.RAO MD 11/16/2012 47 Figure 1.1
  • 48. CLASSIFYING INFECTIOUS DISEASES • Communicable diseases are transmitted directly or indirectly from one host to another. • Chicken pox, genital herpes, • A contagious disease is one that is easily spread from one person to another. • Noncommunicable diseases are caused by microorganisms that normally grow outside the human body and are not transmitted from one host to another • Tetanus, Clostridium tetani DR.T.V.RAO MD 11/16/2012 48
  • 49. THE MODERN AGE OF MICROBIOLOGY DR.T.V.RAO MD 11/16/2012 49
  • 50. THE MODERN AGE OF MICROBIOLOGY • Microbial Genetics • Avery, MacLeod, and McCarty determined genes are contained in molecules of DNA • Beadle and Tatum established that a gene’s activity is related to protein function • Translation of genetic information into protein explained • Rates and mechanisms of genetic mutation investigated • Control of genetic expression by cells described DR.T.V.RAO MD 11/16/2012 50
  • 51. THE MODERN AGE OF MICROBIOLOGY • Molecular Biology • Explanation of cell function at the molecular level • Genome sequencing • Pauling proposed that gene sequences could • Provide understanding of evolutionary relationships and processes • Establish taxonomic categories that reflect these relationships • Identify existence of microbes that have never been cultured • Woese determined that cells belong to bacteria, archaea, or eukaryotes • Cat-scratch fever caused by unculturable organism DR.T.V.RAO MD 11/16/2012 51
  • 52. THE MODERN AGE OF MICROBIOLOGY • Recombinant DNA Technology • Genes in microbes, plants, and animals manipulated for practical applications • Production of human blood- clotting factor by E. coli to aid hemophiliacs • Gene Therapy • Inserting a missing gene or repairing a defective one in humans by inserting desired gene into host cells DR.T.V.RAO MD 11/16/2012 52
  • 53. DISCOVERY OF ANTIMICROBIAL AGENTS _________  Alexander Fleming (1881 – 1955), a Scottish biologist and pharmacologist, observed bacterial staphylococci colonies disappearing on plates contaminated with mold.  Fleming extracted the compound from the mold responsible for destruction of the bacterial colonies.  The product of the mold was named penicillin, after the Penicillium mold from which it was derived.  Nobel Prize in Physiology of Medicine in 1945. Images: Penicillium mold, PHIL #8396; Staphylococcus aureus on DR.T.V.RAO MD 11/16/2012 53 antibiotic test plate, PHIL #2641; Poster attached to a mailbox From the Virtual Microbiology Classroom on ScienceProfOnline.com offering advice to World War II servicemen, 1944, NIH
  • 54. THE MODERN AGE OF MICROBIOLOGY • How Do We Defend Against Disease? • Serology • The study of blood serum • Von Behring and Kitasato – existence in the blood of chemicals and cells that fight infection • Immunology • The study of the body’s defense against specific pathogens • Chemotherapy • Fleming discovered penicillin • Domagk discovered sulfa drugs DR.T.V.RAO MD 11/16/2012 54
  • 55. THE BIRTH OF MODERN CHEMOTHERAPY • Treatment with chemicals is chemotherapy. • Chemotherapeutic agents used to treat infectious disease can be synthetic drugs or antibiotics. • Antibiotics are chemicals produced by bacteria and fungi that inhibit or kill other microbes. • Quinine from tree bark was long used to treat malaria. • 1910: Paul Ehrlich developed a synthetic arsenic drug, salvarsan, to treat syphilis. • 1930s: Sulfonamides were synthesized. DR.T.V.RAO MD 11/16/2012 55
  • 56. THE BIRTH OF MODERN CHEMOTHERAPY • 1928: Alexander Fleming discovered the first antibiotic. • He observed that Penicillium fungus made an antibiotic, penicillin, that killed S. aureus. • 1940s: Penicillin was tested clinically and mass produced. DR.T.V.RAO MD 11/16/2012 56
  • 57. MODERN DEVELOPMENTS IN MICROBIOLOGY • Immunology is the study of immunity. Vaccines and interferons are being investigated to prevent and cure viral diseases. • The use of immunology to identify some bacteria according to serotypes (variants within a species) was proposed by Rebecca Lancefield in 1933. DR.T.V.RAO MD 11/16/2012 57 Figure 1.4 (3 of 3)
  • 58. MODERN BIOTECHNOLOGY AND GENETIC ENGINEERING • Biotechnology, the use of microbes to produce foods and chemicals, is centuries old. • Genetic engineering is a new technique for biotechnology. Through genetic engineering, bacteria and fungi can produce a variety of proteins including vaccines and enzymes. DR.T.V.RAO MD 11/16/2012 58
  • 59. SELECTED NOBEL PRIZES IN PHYSIOLOGY OR MEDICINE 1901* von Behring Diphtheria antitoxin 1902 Ross Malaria transmission 1905 Koch TB bacterium 1908 Metchnikoff Phagocytes 1945 Fleming, Chain, Florey Penicillin 1952 Waksman Streptomycin 1969 Delbrück, Hershey, Luria Viral replication 1987 Tonegawa Antibody genetics . 1997 Prusiner Prions DR.T.V.RAO MD 11/16/2012 59
  • 60. SELECTED NOVEL PRIZES IN PHYSIOLOGY OR MEDICINE 1901* von Behring Diphtheria antitoxin 1902 Ross Malaria transmission 1905 Koch TB bacterium 1908 Metchnikoff Phagocytes 1945 Fleming, Chain, Florey Penicillin 1952 Waksman Streptomycin 1969 Delbrück, Hershey, Luria Viral replication 1987 Tonegawa Antibody genetics 1997Prusiner Prions 2003Agre, Mackirron water and ion channels 2005 Marshall, Warren Helicobacter and ulcers 2008 Hausen Papilloma and viruses * The first Nobel Prize in Physiology or Medicine. DR.T.V.RAO MD 11/16/2012 60
  • 61. UNIVERSAL PRECAUTIONS SET UP BY CDC • Use gloves, gowns, masks and goggles • Minimize risk of needle sticks • Disinfections procedure • Preventative treatment after exposure • Reduce risk • Treat all patients the same • HBV greater risk than HIV DR.T.V.RAO MD 11/16/2012 61
  • 62. DEAR STUDENTS NEVER FORGET TO WASH HANDS AFTER HANDLING PATIENTS OR INFECTED MATERIAL DR.T.V.RAO MD 11/16/2012 62
  • 63. VISIT ME FOR MORE ARTICLES OF INTEREST ON MICROBIOLOGY, INFECTIOUS DISEASES… DR.T.V.RAO MD 11/16/2012 63
  • 64. • Programme Created by Dr.T.V.Rao MD for Undergraduate Medical and Paramedical Students for orientation in Learning Medical Microbiology • email • doctortvrao@gmail.com DR.T.V.RAO MD 11/16/2012 64