Hernandez tops micropathlec exam

Bactol in micropathlab

Sobrevega in physiolec

bug investigators

hard-working micro students

miss evidente evidently doing 'distance learning'

bacterial cell lec slide

Prokaryotes (Bacteria)
• Eubacter "True" bacteria
– human pathogens
– clinical or environmental
– one kingdom
• Archaea
– Environmental organisms
– second kingdom
Eukaryotes
• Other cell-based life e.g.
– plants
– animals
– fungi
Prokaryotic Cell (versus Eukaryotic Cell
• Not compartmentalized
• Cell membranes lack sterols (e.g. cholesterol)
• Single circular chromosome
• Ribosomal are 70S

Bacteria
• Plasmids
• Extra-chromosomal DNA
• multiple copy number
• coding pathogenesis and antibiotic
resistance factors
• bacterial replication
The Cell Envelope
Oxidative phosphorylation occurs at cell membrane
(since there are no mitochondria
The ceined: caA‪yer or glycocalyx
• usually polysaccharide
• often lost on in vitro culture
• protective in vivo
• Many bacterial cells secrete some extracellular material in the form of a capsule or a slime layer
• A slime layer is loosely associated with the bacterium and can be easily washed off

• whereas a capsule is attached tightly to the bacterium and has definite boundaries
Endospores (spores
• Dormant cell
• Produced when starved
• Resistant to adverse conditions
- high temperatures
- organic solvents
• Bacillus and Clostridium

Culture and identification of infectious agents
Taxonomy
• Defines common traits among strains for a bacterial species

• Usually genetic

• Allows development of diagnostic kits
TAXONOMY
Classification, Nomenclature, Laboratory Identification
Identification of infectious agents
in the diagnostic laboratory
• Aids treatment
• Helps antibiotic selection
• General hospital laboratory
– physiological tests
• Reference laboratories
– Genetic tests
Steps in isolation and identification
• Step 1. Streaking culture plates
�A‪yer or glycocalyx
• usually polysaccharide
• often lost on in vitro culture
• protective in vivo
• Many bacterial cells secrete some extracellular material in the form of a capsule or a slime layer
• A slime layer is loosely associated with the bacterium and can be easily washed off

• whereas a capsule is attached tightly to the bacterium and has definite boundaries
Endospores (spores
• Dormant cell
• Produced when starved
• Resistant to adverse conditions
- high temperatures
- organic solvents
• Bacillus and Clostridium

Culture and identification of infectious agents
Taxonomy
• Defines common traits among strains for a bacterial species

• Usually genetic

• Allows development of diagnostic kits
TAXONOMY
Classification, Nomenclature, Laboratory Identification
Identification of infectious agents
in the diagnostic laboratory
• Aids treatment
• Helps antibiotic selection
• General hospital laboratory
– physiological tests
• Reference laboratories
– Genetic tests
Steps in isolation and identification
• Step 1. Streaking culture plates
– colonies on incubation (e.g 24 hr)
– size, texture, color, hemolysis
– oxygen requirement
Samples of body fliuds (blood,urine,csf)
• Step 2. Colonies Gram stained
– cells observed microscopically
Gram stain morphology
• Shape
– cocci (round)
– bacilli (rods)
– spiral or curved (e.g. spirochetes)

• Single or multiple cells
– clusters (e.g. staphylococci)
– chains (e.g. streptococci)

• Gram positive or negative

Step 3. Isolated
bacteria are speciated
• Generally using physiological tests
Step 4.
Antibiotic susceptibility testing
Molecular differentiation
• Genomics

• Gene characterization
– Sequencing
– PCR

• Hybridization

• % guanine + cytosine
16S rRNA Sequencing
• Differentiates bacterial species
• Development of clinical tests based on sequence (e.g. PCR)

Rapid diagnosis without culture
• WHEN AND WHY?
• grow poorly
• can not be cultured
Microscopy
• spinal fluids (meningitis)
• sputum (tuberculosis)

• sensitivity poor
Serologic identification
• antibody response to the infecting agent

• several weeks after an infection has occurred

Nutrition, Growth and Metabolism
Bacterial requirements for growth
• oxygen (or absence)
• energy
• nutrients
• optimal temperature
• optimal pH
Respiration
• Respiration is the oxidation of a source of energy by removal of electrons and donation to an inorganic terminal electron accepter.
Fermentation
• Fermentation is defined as an energy yielding process whereby organic molecules serve as both electron donors and electron accepters

• The molecule being metabolized does not have all its potential energy extracted from it. In other words, it is not completely oxidized
Oxygen Requirements
Obligate aerobes must grow in the presence of oxygen they cannot carry out fermentation
Obligate anaerobes do not carry out oxidative phosphorylation they are killed by oxygen
Aerotolerant anaerobes are bacteria that respire anaerobically but can survive in the presence of oxygen
Facultative anaerobes can perform both fermentation and aerobic respiration
Microaerophilic bacteria grow well in low concentrations of oxygen,but are killed by higher concentrations.
Obligate aerobes
• grow in presence of oxygen
• no fermentation
• oxidative phosphorylation
Obligate anaerobes
• no oxidative phosphorylation
• fermentation
• killed by oxygen
• lack certain enzymes
superoxide dismutase
O2-+2H+ H2O2
catalase
H2O2 H20 + O2
peroxidase
H2O2 H20 /NAD NADH
Facultative anaerobes
• fermentation
• aerobic respiration
• survive in oxygen
Microaerophilic bacteria
• grow
– low oxygen
• killed
– high oxygen

Optimal growth temperature
• Mesophiles:
– human body temperature
* pathogens
* opportunists
• pyschrophile
– close to freezing
• thermophile
– close to boiling
pH
• Many grow best at neutral pH
• Some can survive/grow
- acid
- alkali
Nutrient Requirements
• Carbon
• Nitrogen
• Phosphorus
• Sulfur
• Metal ions (e.g. iron)

Siderophores (S)
Small molecule secreted by bacteria that bind iron
Measuring bacterial mass in liquid cultures of bacteria
Common methods include:
a) turbidity (the cloudiness of a liquid culture of bacteria –
a measure of total bacteria [live and dead]
b) the number of viable bacteria in a culture –
usually assessed by counting the number of colonies
that grow after streaking a known volume on a plate
Measuring bacterial mass (live + dead) in liquid culture
Measured with spectrophotometer
Measuring viable bacteria
Colony forming units

SUGAR CATABOLISM
• Glycolysis
– Embden Meyerhof Parnas Pathway
– most bacteria
– also animals and plants

STERILIZATION
• All killed
• non-selective
• autoclaving
• 121oC (heat/pressure)
* Heat resistant materials
• ethylene oxide
• Non heat resistant
– usually equipment
• ultra-violet light
– surfaces (e.g operating rooms)
not totally effective
• gamma radiation
– food
– some mail
Disinfection
• Liquids that kill bacteria
– e.g. phenol based
– too toxic for skin surfaces
Antiseptics
• Topical (e.g. skin)
– e.g. iodine or 70% alcohol
– “reduce” bacterial load
ANTIBIOTICS
• Selectively toxic for bacteria
– bactericidal (killing)
– bacteriostatic (growth inhibition)

• no harm to patient
• destroy structures
• present in bacteria
• not present in host

Antibiotics work together
with immune system
Minimal inhibitory concentration
• lowest level stopping growth

• e. g. zone of inhibition around a disk impregnated with antibiotic

• Antibiotics that inhibit cell wall
biosynthesis are bactericidal

• Without cell wall, osmotic pressure
causes bacteria to burst

Antibiotics: Protein Synthesis, Nucleic Acid Synthesis and Metabolism
Principles and Definitions
• Selectivity
– Selectivty8 toxicity9
• Therapeutic index
– Toxic dose/ Effective dose
• Categories of antibiotics
– Bactericidal
• Usually antibiotic of choice
– Bacteriostatic
• Duration of treatment sufficient for host defenses
• Categories of antibiotics
– Use of bacteriostatic vs bactericidal antibiotic
• Therapeutic index better for bacteriostatic antibiotic
• Resistance to bactericidal antibiotic
• Protein toxin mediates disease – use bacteriostatic protein synthesis inhibitor
• Antibiotic susceptibility testing (in vitro)
– Minimum inhibitory concentration (MIC)
• Lowest concentration that results in inhibition of visible growth
– Minimum bactericidal concentration (MBC)
• Lowest concentration that kills 99.9% of the original inoculum
• Combination therapy
– Prevent emergence of resistant strains
– Temporary treatment until diagnosis is made
– Antibiotic synergism
• Penicillins and aminoglycosides
• CAUTION: Antibiotic antagonism
• Penicillins and bacteriostatic antibiotics

Antibiotics that Inhibit Protein Synthesis
Protein Synthesis Inhibitors
• Mostly bacteriostatic
• Selectivity due to differences in prokaryotic and eukaryotic ribosomes
• Some toxicity - eukaryotic 70S ribosomes
Inhibitors of Nucleic Acid Synthesis
• Rifampin, Rifamycin, Rifampicin, Rifabutin (bactericidal
• Quinolones (bactericidal)
nalidixic acid, ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, lomefloxacin, sparfloxacin
Antimetabolite Antimicrobials
• Inhibitors of Folic Acid Synthesis

• Sulfonamides, Sulfones (bacteriostatic)
• Trimethoprim, Methotrexate, Pyrimethamine (bacteriostatic)
Antimicrobial Drug Resistance
Principles and Definitions
• Clinical resistance
• Resistance can arise by mutation or by gene transfer (e.g. acquisition of a plasmid)
• Resistance provides a selective advantage
• Resistance can result from single or multiple steps
• Cross resistance vs multiple resistance
– Cross resistance -- Single mechanism-- closely related antibiotics
– Multiple resistance -- Multiple mechanisms -- unrelated antibiotics
Antimicrobial Drug Resistance Mechanisms
• Altered permeability
– Altered influx
• Gram negative bacteria
– Altered efflux
• tetracycline
• Inactivation
– $-lactamse
– Chloramphenicol acetyl transferase
• Altered target site
– Penicillin binding proteins (penicillins)
– RNA polymerase (rifampin)
– 30S ribosome (streptomycin)
• Replacement of a sensitive pathway
– Acquisition of a resistant enzyme (sulfonamides, trimethoprim)
Pathogenicity
• virulence factors
• number of initial organisms
• immune status
Koch's postulates
• isolated
– diseased not healthy people
• growth
– pure culture
• induce disease
– susceptible animals
• re-isolated
– susceptible animals

Opportunistic infections
• compromised people
– normal flora
– environment

Opportunists - normal flora
• Skin
– Staphylococcus aureus,
– S. epidermidis
– Propionibacterium acnes
• Intestine
– Bacteroides
* high numbers
– Enterobacteriaceae
* low number
Opportunists - environment
» air
• water
• soil
• food
• Opportunists in hospital- nosocomial

Transmission
• airborne droplets
• food
• water
• sexual contact
Host defenses
• Gut
– peristalsis
– defecation
• respiratory tract
– ciliary action
– coughing
– sneezing
• urogenital tract
– urination
COMMENSALS CAUSE DISEASE
WHEN HOSTS DEFENCES ARE LOW
PATHOGENS POSSESS VIRULENCE FACTORS
THAT ENABLE THEM TO CAUSE DISEASE IN
THE PRESENCE OF NORMAL DEFENCES
DETERMINANTS OF BACTERIAL PATHOGENISIS
TRANSMISSION
INFECTING DOSE-INOCULUM
PORTAL OF ENTRY
BACTERIAL VIRULENCE FACTORS
CAPSULES
INVASIVENESS
ADHESINS
EXOENZYMES
TOXINS
CAPSULES
LOCATED EXTERNAL TO THE CELLWALL
COMMON VIRULENCE FACTOR
POLYSACCHARIDES
AVOID OR SURVIVE PHAGOCYTOSIS
ADHESINS
ALLOW BACTERIA TO STICK ON MUCOSAL SURFACE
ADHESINS ARE SURFACE FACTORS
PILI / FIMBRIAE
LPS LIPOPOLYSACCARIDE SIDECHAINS
M PROTEIN
INVASIVENESS
ABILITY TO INVADE HOST CELL
MULTIFACTORIAL COMPLEX PROCESS
MAY INCLUDE ADHESION AND ENZYMES FACTORS

EXOENZYMES
PRODUCE AND SECRETED BY BACTERIA
ENZYMES THAT BREAKDOWN COLLAGEN AND FIBRIN
COLLAGENASES / HYALURONIDASES / FIBRINOLYSINS
ENZYMES THAT BREAKDOWN CELLULAR MATERIALS
PROTEASES / LECITHINASES
ENZYMES THAT DEACTIVATE AND MODIFY ANTIBIOTICS
BETA LACTAMASE
ENZYMES THAT DESTROY NEUTROPHILS
AND MACROPHAGES
LEUKOCIDINE
ENZYMES THAT ACCELERATE FIBRIN CLOT
COAGULASE
TOXINS
2 CLASSIFICATIONS
EXOTOXINS – PROTEINS PRODUCED AND
SECRETED BY BACTERIA
ENDOTOXINS – PART OF THE BACTERIA ITSELF
EXOTOXIN
NEUROTOXINS- AFFECT NERVOUS TISSUE
TETANUS TOXIN
ENTEROTOXINS – AFFECT GASTROINTESTINAL TRACT
CHOLERA TOXIN
CYTOTOXINS – AFFECT CELLS OF VARIOUS TISSUES
DIPHTERIA TOXIN
ENDOTOXINS
ALL GM NEGATIVE BATERIA HAVE ENDOTOXINS
IN OUTER MEMBRANE
RELEASED AS BACTERIA ARE LYSED
RESPONSIBLE FOR SEPSIS AND
SEPTIC SHOCK - FATAL

ectron transport chain of proteins

synthesis of ATP is a series of reduction-oxidation(redox) reaction..
watch and learn by reviewing the questions