Microbiology: Systematic Bacteriology

1. Appearance/Structure
   1. 5 features
      1. Shape
         1. Cocci
            1. Sphere shaped
            2. Diplococcus
               1. Divides in one to procduce two cocci
            3. Gram Negative
            4. Gram Positive
               1. Aerotolerant
                  1. Streptococcus
                     1. Divides in one plane to produce CHAINS of 4 - 20
                     2. Growth is indifferent to oxygen level
                     3. Streptococcus pneumoniae

                        Annotations:

                        • Gram positive alpha haemolytic cocci. Part of the normal upper respiratory tract flora in many people. Commonest cause of pneumonia Also causes severe form meningitis.
                        • 90+ different capsular types - vaccines available against the commonest 23 types  Majority of UK strains are sensitive to penicillin.
                        1. Commonest cause of pneumonia

                           Annotations:

                           • Pneumonia is an acute inflammation of the lungs often caused by pneumococci of the species streptococcus pneumonia. The alveoli and bronchioles of the lung becomes plugged with a fibrous exudate.
                        2. Also causes severe form meningitis
                        3. Vaccines available

                           Annotations:

                           • Against commonest 23 types of streptococcus pneumonia.
                     4. Found in dental plaque of most mammalian species
                     5. Classified using haemolysis
                     6. GAS
                        1. Group A Streptoccoci
                           1. SIGNIFICANT PATHOGENIC STREPTOCOCCI
                           2. Causes various diseases including
                              1. Streptococcal sore throat (scarlet fever)
                              2. Invasive diseases
                                 1. Necrotising fasciitis
                              3. Puerperal sepsis
                                 1. Infection of pregnant and post natal women
               2. Aerobic
                  1. Enterococcus
                     1. Causes enteric infections
                        1. Enterococcus faecalis
                           1. Not particularly pathogenic
                        2. Enterococcus faecium
                           1. Part of normal bowel flora
                           2. Cause problems if they get into a normally sterile site
                              1. These are commonest cause of UTIs
                     2. MOST IMPORTANT GROUP OF NON-HAEMOLYTIC STREPTOCOCCI
               3. Facutative aerobes

                  Annotations:

                  • They are versatile organisms,  capable of growth under both aerobic and anaerobic conditions. They preferentially use oxygen as terminal electron acceptor.
                  1. Staphylococcus

                     Annotations:

                     • Gram positive non-motile bacteria that form irregular grape-like clusters.
                     1. Divides in three planes to produce clumps

                        Annotations:

                        • Forms irregular grape-like clusters.
                     2. Infection sources are nosocomial and community
                        1. Nosocominal
                           1. (of a disease) originating in a hospital.
                           2. Types of infection associated with foreign devices
                              1. For example
                                 1. Catheter
                           3. MRSA
                              1. Mainly nosocomial
                              2. More commonly affects the elderly and immunocompromised
                                 1. Individuals:
                                    1. In ICU
                                    2. Burns
                                    3. Dialysis
                                    4. Surgical patients
                                    5. With intravenous lines
                     3. Non-motile bacteria
                     4. S.aureus
                        1. Commonest cause of skin, soft tissue and wound infection
               4. Anaerobic
                  1. Clostridium ssp
                     1. Produces spores
                     2. Clostridium difficile
                        1. Opportunistic pathogen
                           1. Proliferates in the absence of normal flora
                        2. Part of the normal flora of man and animals
                        3. Produces endotoxins that cause severe tissue damage
                        4. Pseudomembranous colitis

                           Annotations:

                           • Swelling or inflammation of the large intestine (colon) due to an overgrowth of clostridium difficile bacteria.
                           1. Causes antibiotic associated diarrhoea in the elderly

                              Annotations:

                              • Occurs as a result of pseudomembranous colitis (mild to severe 2 days to 6 weeks after antibiotic treatment.
                        5. Endemic could occur
                           1. Strains are producing more toxins
                           2. Antibiotic resistance
                           3. Increased sporulation
                     3. Clostridium perfringens
                        1. Causes "gas" gangrene

                           Annotations:

                           • A severe soft tissue infection following contamination of wound.
                     4. Clostridium tetanus
                        1. Causes tetanus

                           Annotations:

                           • Tetanus is a usually fatal paralytic illness.
         2. Bacilli
            1. Rod shaped
            2. Can form chains
            3. May be slightly curved
               1. For example vibrio (gram negative bacteria)
            4. Fusiform bacteria

               Annotations:

               • Small bacteria that are spindle-shaped rods.
               1. F.nucleatum
                  1. Found in the dental plaque of many promates
            5. Gram negative
               1. Strict aerobes
                  1. Pseudomonas aeruginosa
                  2. Legionella pneumophila
                     1. Causes Legionnaires disease
                        1. Usually environmental chest infection
               2. Anaerobe
                  1. Vibrio cholera
                     1. Causes cholera

                        Annotations:

                        • A severe diarrhoeal illness.
               3. Microaerophilic

                  Annotations:

                  • Requires low but not full oxygen tension.
                  1. Campylobacter ssp
                     1. Spiral shaped
                     2. Commonest cause of bacterial diarrhoea in the UK
                  2. Helicobacter spp
                     1. Infects the stomach
                        1. Causes gastritis
                        2. Causes duodenal ulcers
               4. Capnophiles

                  Annotations:

                  • Capnophilic bacteria require increased concentration of carbon dioxide (5% to 10%) and approximately 15% oxygen. This condition can be achieved by a candle jar (3% carbon dioxide) or carbon dioxide incubator, jar or bags.
                  1. Haemophilus influenzae
                     1. Common cause of chest infection
                     2. Controlled by vaccination
                     3. Cocco bacillus
            6. Gram positive
         3. Spiral shaped

            Annotations:

            • Spiral shaped bacteria can be gram positive or gram negative.
            1. 2 main types
               1. Rigid
                  1. For example Spirillum
               2. Flexible
                  1. Spirochaete
      2. Size
      3. Arragement
      4. Structures
         1. Capsules

            Annotations:

            • A polysaccharide layer that lies outside the cell envelope of bacteria and is thus deemed part of the outer envelope of a bacterial wall. It is a well-organised layer, not easily washed off and it can be the cause of various diseases.
            1. Polysaccharide layer
               1. Lies outside the cell envelope of bacteria
               2. Part of the OUTER envelope of a bacterial cell
               3. Well organised
               4. Not easily washed off
               5. Can cause various diseases
               6. THIS IS NOT THE 2nd LIPID MEMBRANE LATER OF GM -VE BACTERIA
            2. Found on both Gram Negative and Gram positive bacteria
         2. Flagella
         3. Spores
            1. Inert structures
               1. Resistant to physical and chemical challenge
            2. For example C.difficile produces spores
            3. Spore formation
               1. BINARY FISSION
                  1. SPORULATION
                     1. PRESPORE
                        1. ENDOSPORE
                           1. SPORE
                              1. GERMINATION
                                 1. The development of a spore after a period of dormancy
                              2. Spore-like structure produced by bacteria
                           2. A dormant, tough, and non-reproductive structure produced by certain bacteria
                        2. Cells that later differentiate into spores are then formed
                     2. Formation of spores (the spontaneous division of a cell into four or more daughter cells, each of which contains a part of the original nucleus.
                  2. Method of asexual reproduction in whihc an organism splits in two
         4. Pili

            Annotations:

            • This term is interchangeable with fimbria
            1. AKA Fimbria
            2. Hair like appendage
            3. Found on the surface of many bacteria
      5. Cell wall
         1. Gram positive
            1. Examples of common gram positive infectious bacteria in the UK include:
               1. Streptococcus ssp
               2. Staphylococcus ssp
               3. Enterococcus ssp
               4. Clostridium
         2. Gram Negative
            1. Examples of common gram negative infectious bacteria in the UK include:
               1. Neisseria ssp
                  1. Neisseria meningitidis
                     1. Commonest cause of bacterial meningitis
                  2. Neisseria Gonorrhoeae
                     1. Causes gonorrhoea
               2. Echierichia ssp
                  1. Verotoxin (VTEC) producing Escherichia coli
                     1. E.coli O157
                     2. E.coli O104
                     3. SIGNIFICANT GUT PATHOGEN
               3. Salmonella ssp
                  1. SIGNAFICANT GUT PATHOGEN
               4. Shigella ssp
                  1. SIGNIFACENT GUT PATHOGEN
            2. Aerobic gram negative cocci
               1. Appear in pairs on gram film
                  1. Diplococci
            3. Coliform

               Annotations:

               • pertaining to fermentative gram-negative enteric bacilli, sometimes restricted to those fermenting lactose, i.e., Escherichia, Klebsiella, Enterobacter, and Citrobacter.
               1. Gram negative bacilli
               2. Sometimes restricted to describing lactose fermenting gram negative bacilli
                  1. For example:
                     1. Escherichia
                     2. Klebsiella
                     3. Enterobacter
                     4. Citrobacter
               3. Can also grow anaerobically
               4. Grow best in aerobic environment
               5. Differentiated from each other by:
                  1. Biochemical reactions
                  2. Antigenic structure of cell wall

                     Annotations:

                     • The presence of O antigens on the cell wall and H antigens on the flagella of a bacteria are examples of antigens that can be used to distinguish between coliform.
                     1. Serotyping
               6. If these enter a normally sterile site:
                  1. CAN CAUSE SERIOUS INFECTION
                     1. For example:
                        1. UTI
                        2. Peritonitis
                        3. Bilary tract infectoin
                     2. Patients with coliform sepsis
                        1. Become VERY unwell VERY quickly
                           1. Due to endotoxin
                              1. This is released from the cell wall of gram negative bacteria when they die
                              2. Endotoxin shock
                                 1. SIRS
                                    1. Systemic Inflammatory Response Syndrome
               7. GENTAMICIN
                  1. 1st line of antibiotic used to treat infections caused by coliforms
         3. Gram stain
            1. Distinguishes 2 major classes of bacteria according to cell wall structure
            2. Procedure
               1. 1)Heat fixed film of bacteria on a glass slide
               2. 2) Stain with CRYSTAL VIOLET for 1 minute and rinse off with water
               3. 3) Treat with Gram's iodine for 1 minute then rinse with water
               4. 4) Briefly decolourise with acetone or ethanol
                  1. A few seconds depending on thickness of film
                  2. Bacteria changes colour
                     1. BLACK TO PURPLE
                        1. GRAM POSITIVE BACTERIA
                     2. BLACK TO COLOURLESS
                        1. GRAM NEGATIVE BACTERIA
                  3. 5) Counter stain with basic fuchsin or safranin (pink dye) for 1 minute and tinse with water
                     1. GRAM POSITIVE BACTERIA WILL REMAIN PURPLE
                     2. GRAM NEGATIVE BACTERIA WILL CHANGE FROM COLOURLESS TO PINK
                     3. 6) Blot dry and view under oil emersion
            3. Has limitations
               1. There are:
                  1. Gram variable bacteria and bacteria that do not stain with crystal violet/iodine complex

                     Annotations:

                     • Examples of gram variable bacteria include mycobacterium tuberculosis and teponema pallidum (causes syphilis)
2. Growth requirements
   1. Used to categorise bacteria
   2. Aerobic/Anaerobic

      Annotations:

      • Aerobic bacteria grow in oxygen/air
      1. Strict anaerobes
      2. Strict aerobes
      3. Obligate anerobes
         1. Bacteria which are killed by oxygen
         2. Respiration uses electron acceptor other than oxygen
         3. Smaller reduction potential than O2
         4. Less protein motive force across membrane (ATP synthase)
         5. Less energy released per moleule oxidised
      4. Obligate aerobes
         1. Bacteria which require oxygen
      5. Fascilitative bacteria
         1. Bacteria which tolerate oxygen
   3. Requirement for blood products
      1. Serum proteins
   4. Sensitivity to inhibitory agents
      1. NaCl
      2. Bile
      3. K tellurite
   5. Media
      1. Bacteria are grown in selective media

         Annotations:

         • Selective media: concept that the presence of specific substances permits the growth of one organism over the other.
         1. For example
            1. Mannitol salt agar (MSS): 7.5% salt allows preferential isonaltion of staphylococci
            2. Salmonella-Shingella (SS): Bile salts inhibit coliforms
      2. Differential media

         Annotations:

         • Incorporation of chemicals produces visible change colonies that FASCILITATE IDENTIFICATION (differentiation)
         1. MacConkay Agar
            1. Fascilitates identification of enterobacteriacecae
            2. There are several types of blood agar including:
               1. MacConkey
               2. CBA
                  1. Cooked Blood Agar
                     1. Contains V factor

                        Annotations:

                        • Required by some organisms for growth (complex nutritional requirement).
                        1. This is trapped inside RBCs
                     2. Heated then cooled to 50 degrees celsius in an autoclave
               3. Chocolate agar

                  Annotations:

                  • A type of blood agar in whihchthe blood cells have been lysed by heating for growing fastidious organisms.
                  1. Contains V factor

                     Annotations:

                     • Required by some organisms for growth (complex nutritional requirement).
                     1. Released from within erythrocytes
                  2. Blood cells are lysed
                  3. Used to grow fastidious organisms
               4. Contaminant

                  Annotations:

                  • An organism that has gotten into a culture by accident.
         2. EMB
            1. Eosin and methylene blue (EMB)
            2. Allows identification of LACTORSE FERMEMTERS
               1. For example E.coli
               2. Coliform
3. Enzyme/ metabolic tests
   1. 3 features used
      1. Coagulate test
      2. Haemolysis
         1. STREPTOCOCCI ONLY
         2. ALPHA Haemolysis
            1. Bacteria causes INCOMPLETE lysis of red blood cells
               1. Therefore GREENISH zones around their colonies are formed
         3. BETA Haemolysis
            1. Bacteria causes COMPLETE lysis of red blood cells
               1. Therefore they form CLEAR zones around their colonies on the blood agar
            2. GAS
               1. Group A Streptococci
         4. GAMMA Haemolysis
            1. Bacteria cause NO haemolysis of red blood cells
      3. Biochemical profiling
         1. For example carbohydrates are metabolised
   2. Metabolic profiling
      1. Analyses chemical reactions that regulate humans' health

         Annotations:

         • Some specific microbiology tests are directly involved with investigating a patients condition/disease. e.g. Blood culture Urine culture Faeces culture Swab of pus Specimens for polymerase chain rection (PCR tests) Blood serology (Antibody/antigen etc)
         1. Bacteria utilisation of carbon sources and amino acids are analysed
         2. Fluids such as plasma and urine (exo-enzyme production of urease is analysed) are often used to gether the necessary information
4. Molecular tests
   1. 3 features used
      1. Immunological tests
         1. For example cell surface antigens
      2. DNA Sequencing
         1. qPCR
            1. Real Time PCR

               Annotations:

               • The process of detecting amplicon (a piece of DNA) (made possible by the labelling of primer, probes or DNA sections with fluorogenic molecules), enabling the visualisation of accumilating DNA sections as the amplification of a DNA progresses during PCR.
         2. 16sRNA
         3. Multi Locus Sequencing Typing

            Annotations:

            • A technique of molecular biology for the typing of multiple loci. The procedure characterises isolates of mictobial species using DNA sequences of internal fragments of multiple housekeeping genes
            1. MLST
      3. Protein profiling
         1. For example mass spectroscopy analysis
   2. Serological tests

      Annotations:

      • Tests that detect a specific molecule believed to be there (confirms its presence).
      1. Detect a specific molecule believed to be there ( it confirms its presence)
      2. Agglutination

         Annotations:

         • Process of particles clumping that occurs if an antigen is mixed with its corresponding anti-body (called isoagglutinin)
   3. MALDI-TOF

      Annotations:

      • Stands for: MATRIX ASSOCIATED LASER DESORPTION - TIME  OF  FLIGHT
      • Various benefits: Powerful (99% correct) Rapid (Colony tested in 6 minutes) Precise (Species, sub species and sometimes strains can be determined) Cost effective (17 - 32% cost of conventional techniques)
      • - Generates a series of ions from a sample dependent on its constituents - Separates the ion according to mass and charge - Detects the spectrum of proteins released - Results in characteristic signature (mass spectroscopy)
      1. TOF

         Annotations:

         • Charged ions of various sizes are genrated on the sample side. Apotential difference between the sample slide and the ground attracts the ions to the detector.
         1. Velocity of the attracted ions is determined by the LAW OF CONCERVATION OF ENERGY

            Annotations:

            • As the potenial difference V0 is constant with respect to all ions, ions with smaller mass per charge value (lighter ions)  and more highly charged ions move FASTER though the drift space until they reach the detector. Concequently, the time of ion flight differs according to the mass-to-charge ratio (m/z) value of the ion
         2. TIME
         3. OF
         4. FLIGHT
      2. MALDI

         Annotations:

         • The matrix absorbs the ultraviolet light (nitrogen laser light, wavelength 337 nm) and converts it to heat energy. A small part of the martix (down to 100 nm from top outer suface to analyte) heats rapidly (in several nanoseconds) and is vapourised together with the sample.
         1. MATRIX
         2. ASSOCIATED
         3. LASER
         4. DESORPTION
      3. Limitations

         Annotations:

         • Not so good at identifying: STREPTOCOCCI STRAPHYLOCOCCI
         1. Not so good at identifying:
            1. STREPTOCOCCI
            2. STAPYLOCOCCI
      4. Various benefits:
         1. Powerful
         2. Rapid
         3. Precise
         4. ost effective

1. 3 categories
   1. FORM
      1. Punctiform
      2. Circular
      3. Filamentous
      4. Irregular
      5. Rhizoid
      6. Spindle
   2. ELEVATION
      1. Flat
      2. Raised
      3. Convex
      4. Pulvinate
      5. Umbonate
   3. MARGIN
      1. Entire
      2. Undulated
      3. Lobate
      4. Erose
      5. Filamentous
      6. Curled
2. Colony identification
   1. Colony innoculated/resuspended
      1. 100 microlitres is transferred to an automated biochemical profiling machine
         1. Automated biochemical profiling machine offeres a wide range of tests for common substances

1. Manner of development of a disease
2. Pathogens are released/spread
3. This results in a resevoir of pathogens
4. Contact allows entry
5. Pathogens infect host
6. 5 stages
   1. Adhere/colonise and invade
   2. Evade host
   3. Multiply/ Complete its (pathogen) life cycle
   4. Exit host (host is damaged
   5. Release/Spread
7. Pathogen

   Annotations:

   • Harmful organism that produces a pathology.
   1. Harmful organism
   2. Produces a pathology
   3. Opportunistic pathogen

      Annotations:

      • An organism that causes infection when opportunity/change in natural immunity changes (e.g. in an immunocompromised individual)
      1. Causes infection
         1. When environment changes
   4. Transient microbes

      Annotations:

      • Microbes that may attempt to colonise the same areas of the body resistant to microbiota, but are unable to remain in the body for extended periods of time due to: Difficulty competing with established resident microbes Elimination by body's immune system Physical or chemical changes within the body that discourage growth of transient microbes

Annotations:

• Capacity of a microbe to cause damage to the host.

1. Endogenous microorganisms

   Annotations:

   • Microorganisms found externally or internally in the human body that contribute to the natural flora
   1. Found in human body
      1. Internally
      2. Externally
      3. Contribute to natural flora
         1. Commensal

            Annotations:

            • An organism that is part of the normal flora. (Often mutualistic relationship Often endogenous organisms)
2. Exogenous organisms
   1. Not part of the human flora
   2. Includes potential pathogens
3. Microbiome
   1. Totality of:
      1. Microbes
      2. Genetic material of microbes
      3. Environmental interactions
4. Toxins are KEY to virulence

   Annotations:

   • Toxins that have bound to ans been internalised into intestinal epithelial cells (for example) may: Impair the function of intestinal epithelial cells (diarrhoea) Stimulate cytokine release and activation of macrophages and monocytes (inflammation)

Annotations:

• Prokaryotic, single celled organisms

1. Prokaryotic
2. Single celled organisms

1. 4 categories
   1. Infection
      1. May result in:
         1. Fever
            1. Mechanism
               1. 1) Antigen (component of micro-organisms) or lipopolysaccharide (endotoxin) interact with macrophages
               2. 2) Macrophages release CYTOKINES into bloodstream
               3. 3) Cytokines travel to ANVERIOR hypothalamus (brain)
               4. 4) Cytokines also stimulate adverse effects of sepsis
                  1. Prostaglandin E is released, which INCREASES the body's thermal set point

                     Annotations:

                     • Normal core body temperature is 37 degrees Celsius. Body temperature at fever is greater than 38 degrees Celsius Human pathogens grow best at 37 degrees Celsius. (Growth starts to slow if temperature increases).
                     • Raising the core temperature is an ADAPTIVE RESPONSE and is considered to be beneficial in fighting infection. (However harm may result from fever).
                     1. ADAPTIVE RESPONE
               5. The body now perceives that it is cold
                  1. Starts to "shiver"
                     1. Muscle tone increases>>voluntary muscle movement occur
                     2. Increases metabolic ...
                  2. Vasoconstriction of arterioles
                     1. Limit heat loss by readiation
                        1. Conserves heat
            2. Increases survival from infection
   2. Sepsis
      1. Mechanism
         1. 1)Small blood vessels become "leaky" and loose fluid into the tissues
         2. 2)Lower blood volume requires heart to work harder to maintain oxygenation
            1. HEART RATE INCREASES
            2. 3)Poor tissue perfusion
               1. Blood supply to LESS ESSENTIAL organs is shut down to try to maintain blood supply to brain

                  Annotations:

                  • Less essential organs include skin, kidneys and liver.
                  1. 4) Blood clotting system is activated
                     1. This is an attempt to stop LOSS OF BLOOD in blood vessels
                        1. BUT this blood loss has been caused by "leaking" as opposed to wound
                     2. Results in clotting of tiny vessels
                     3. Uses up all clotting factors
                        1. Increased risk of hemorrhage
      2. Host response to SEVERE INFECTION
   3. Severe sepsis
   4. Septic shock