Slide 1
Kochs PostulatesThe organism must be isolated in all cases of a
designated disease and not from healthy animalsThe organism must be
isolated from the infected person (or animal) and grown in pure
cultureThe organisms from the pure culture must reproduce the
disease when inoculated in a susceptible animalThe organism then
must be isolated from the experimentally infected animalCan you
think of some shortcomings?1Robert Koch: German physician, famous
for isolating Vibrio cholerae, Mycobacterium tuberculosis, and
Bacillus anthracis. Koch also had: the organism occurs in no other
disease as a fortuitous and nonpathogenic parasite.The 4th
condition was added later, not emphasized by Koch. Obvious
shortcomingsnot culturable microorganisms can cause disease
(prions). Also, asymptomatic carriage is possible with many
pathogens. Some human pathogens do not infect animals.
Limitations: do not apply to organisms that cannot be grown in
cell-free culture, such as viruses, or organisms that only cause
disease in humans (cannot be given to humans to see if disease
results)
Molecular version using sequence-based detection of
microorganisms has been
developedLipopolysaccharide=lipid+polysaccharide
Saturated fatty acids pack tightly to decrease permeability to
hydrophobic agentsO-antigen required for virulence, protects
against phagocytosis, killing by complementLipid A = endotoxin
2The long O antigens keep complement off the surface of the cell
so that it does not activate and destroy the cell
Endotoxin: within the membrane, not secretedbut comes off with
membrane blebbing
Porin forms trimer in bacterial outer membrane. Pore is ~10A,
and allows passage of H2O, amino acids, monosaccharides,
disaccharides
Monomer (b-barrel) sideview top viewtrimerMay exclude
antibiotics, toxic chemicals, lysozymeContributes to function of
outer membrane as size exclusion barrierPeriplasmic SpaceOnly in
gram negativesA compartmentFilled with gel of peptidoglycan and
waterDegredative enzymes found hereAmylases, nucleasesBreak down
large nutrientsDetoxifying enzymes found hereBeta-lactamases
4Like an extra room in the houseIf the beta lactamases were
secreted into the environment, they would diffuse away, but in the
periplasmic space they concentrate where the organism needs
themCytoplasmic membraneSite of metabolic pathwaysElectron
transport (respiration)Proton gradient used for ATP synthesis,
flagellar motionCell wall biosynthesis (peptidoglycan, LPS)Sensing
the environmentChemotaxis, signal transductionNutrient transport;
Diffusion: passiveFacilitated: specific poreSymporter or
antiporter: exchangeActive transport: uses ATPEfflux of
macromoleculesMultidrug efflux pumps
5http://www.lib.berkeley.edu/BIOS/covers/cover0054.htmlReprinted
from: Yu, Edward W.; Aires, Julio R.; Nikaido, Hiroshi. AcrB
multidrug efflux pump of Escherichia coli: Composite
substrate-binding cavity of exceptional flexibility generates its
extremely wide substrate specificity. Journal of Bacteriology.
185(19). October 2003. 5657-5664 Copyright 2003 with permission
from ASM.
Peritrichous bacteria: flagellae coalesce into a tailduring one
direction of rotation, opposite rotation leads to tumblingBacterial
MetabolismObligate anaerobes: killed by oxygenClostridium
perfringens: gas gangreneObligate aerobes: require
oxygenMycobacterium tuberculosis: lung TBFacultative: can grow with
or without oxygenEscherichia coli
7Aerobic green, pediatric yellow, mycobacteria red, anaerobic
orange
Bottles contain a resin for binding antibioticsAlso contain a
carbon dioxide sensor on the bottomchanges color if bacteria are
present that produce gas, thus triggering a light sensor, that
tells machine to alert lab tech that the bottle is positive. Next
is a gram stain of the infected liquid, then identification and
sensitivity of the organism.Gram Stain and Culture ResultsAnaerobic
cultures sent intraoperatively grew PrevotellaStrict anaerobe
http://pathmicro.med.sc.edu/ghaffar/brucell03-2.jpghttp://www.hull.ac.uk/mouthcare/cal-mouth/mouthcare/images/fig9.jpg
Anaerobes: Clostridium, Bacteroides, Prevotella,
Fusobacterium8This is actually a picture of brucellabut Prevotella
is a small, poorly staining, gram-negative coccobacillary rod.
Clostridia make spores. Bacteroides, Prevotella, Porphyromonas, and
Fusobacterium are medically-important anaerobic gram negative
organisms that do not sporulate.Plasmids and TransposonsB.
Transposons1. Small genetic elements capable of mediating their own
movement from one chromosomal (or plasmid) location to another2.
Compound transposons carry accessory genes, such as antibiotic
resistance genesC. Clinical Relevance1. Mechanism of spread of
antibiotic resistance among gram-positive and gram-negative
bacteria99ConjugationA. Plasmid-mediated process of DNA transfer
from one cell to another that requires:1. Cell-to-cell contact and
formation of a conjugation bridge via a sex pilus2. A conjugal
plasmid capable of transfer replicationB. Plasmid transfer by
conjugation1. Conjugative plasmid in donor (male) cell encodes sex
pili to initiate cell-cell contact2. Transfer replication creates a
single-stranded copy of the plasmid that is transferred to the
recipient (female). Transfer replication provides the driving force
for transfer and is absolutely required for conjugation.3.
Single-stranded plasmid is converted to a circular duplex molecule
in the recipient (now male)
1010
Bacterial ConjugationC. Transfer of chromosomal genes by
conjugation1. Happens when the conjugal plasmid is integrated in
the host cell chromosome2. Transfer replication is initiated within
the plasmid sequences but additional flanking chromosomal DNA is
also conjugated to the recipient3. The transferred DNA cannot be
maintained as an autonomous plasmid so homologous recombination is
required if the incoming DNA is to be maintainedD. Clinical
Relevance1. Spread of antibiotic resistance from plasmid and
chromosomal loci in both gram-positive and gram-negative
bacteria
1212TransformationA. Historical Evidence1. Griffith: avirulent
pneumococcus can be transformed into a virulent bacterium using a
heat-killed extract of the virulent donor strain.2. Avery, MacLeod
and McCarty: transforming principle is DNA, because transformation
is DNAse-sensitive. Transformation does not require cell-to-cell
contact. B. Mechanism1. Development of competence2. DNA binds to
the cell surface3. DNA enters the recipient cell4. DNA integrates
into host chromosome by homologous recombination. Plasmid molecules
can replicate extrachromosomally1313Bacterial TransformationC.
Clinical Relevance1. Examples of capsule exchange among
pneumococci2. Most significant is acquisition of genes involved in
resistance to antibioticsa. Genes that encode antibiotic binding
proteins (e.g., penicillin binding proteins, gyrase/topoisomerase
IV binding proteins)b. Genes that encode enzymes that inactivate
antibiotics (e.g., -lactamase)c. Genes that encode exporters of
antibiotics (e.g., tetracycline efflux)1414Transduction lytics vs
lysogenicD. Transduction1. Result of occasional packaging of host
DNA during lytic infection and subsequent transfer to a recipient
via phage injection2. Steps in the transduction processa. Infection
of donor strainb. Donor DNA packagingc. Infection of recipientd.
Recombination of donor DNA with the recipient chromosomeE. Clinical
Relevance1. Toxin genes can be carried on bacteriophage, e. g. a.
Diphtheria toxin in Corynebacterium diphtheriae b. Streptococcal
erythrogenic toxinc. Botulinum toxin (Clostridium botulinum)d.
Cholera toxin (Vibrio cholerae)1515
Mechanisms Conferring Resistance1616
