Microbiology Flashcards
what are examples of bacteria and viruses, from largest (30μm) to smallest (less than 1μm)
trypanosoma gambiense entamoeba histolytica borrellia recurrentis treponema pallidum bacillus anthracis escherichia coli staphylococcus aureus rotavirus chlamydia trachomatis vaccinia virus influenza virus wart virus poliovirus
what is bacterial nomenclature? what is an example?
staphylococcus (genus) aureus (species)
what is a pathogen?
organism that causes or is capable of causing disease
what is a commensal?
organism which colonises the host but causes no disease in normal circumstances
what is an opportunist pathogen?
microbe that only causes disease if host defences are compromised
what is virulence/pathogenicity?
the degree to which a given organism is pathogenic
what is asymptomatic carriage?
when a pathogen is carried harmlessly at a tissue site where it causes no disease
what is the diameter of the smallest body that can be resolved and seen clearly with the naked eye?
100μm
what are colonies?
bacteria and fungi form macroscopically visible structures comprising at least 10^8 cells (when propagated on solid media)
what are the three domains of life?
bacteria, archaea and eukarya
what is the anatomy of a bacterial cell?
- cytoplasm bound peripherally by a very thin, elastic and semi-permeable cytoplasmic/plasma membrane (phospholipid bilayer)
- the cell wall is outside and closely covering the membrane, which is porous and permeable
- cell division mediated by actin-like protein, FtsZ
- septum/cross wall
- S-layer is a variably demonstrated ordered protein layer
- capsule
- fimbriae
- flagellum
- sex pili
what combines to produce the cell morphology and characteristic patterns of arrangement?
the exact pattern of cell division and the structures associated with the cell wall and cytoplasmic membrane (collectively the cell envelope)
what are the two basic shapes of bacterial cells?
coccus: spherical
bacillus: rod shaped
what shape are bacterial cocci?
spherical
what shape are bacterial bacilli?
rod shaped
what are variations of bacilli?
vibrio: comma shaped
spirillum and spirochaete: spiral
filamentous
what are the shapes and characteristic groupings of various bacterial cells?
- diplococci
- streptococci
- staphylococci
- sarcina
- bacillus
- vibrio
- spirillum
- actinomyces
what are the structures in bacteria cytoplasm that correspond to eukaryotic actin, tubulin and intermediate filaments?
MreB, FtsZ and crescentin respectively
what types of larger structures occur in some species of bacteria under specific growth conditions?
spores or inclusion bodies of storage products e.g. volutin (polyphosphate), lipid (poly-beta-hydroxyalkanoate or triacylglycerol), glucogen or starch
what is a capsule?
protective gelatinous covering layer outside the bacterial cell wall
what is a microcapsule?
a bacterial capsule that is too thin to be resolved with the light microscope
what is loose slime?
soluble large-molecular material that may be dispersed by the bacterium into the environment
what are flagella?
- protein-based filamentous appendages that protrude outwards from the cell wall of bacteria
- organs of locomotion
what are fimbriae/pili?
- hair like structures on bacteria
- mediate adhesion via specific receptor-ligand interactions at their tip
how do bacteria store/package DNA?
- genetic info is mostly contained in a single, long molecule of double-stranded DNA
- DNA can be extracted in the form of a closed circular thread about 1 mm long
- condenses and loops macromolecule into a supercoiled state
what are plasmids?
additional fragments of episomal (extrachromosomal) DNA contained in the bacterium
how many alleles of each gene do bacteria have?
- bacteria are essentially haploid organisms with only one allele of each gene per cell
- may be multiple copies of chromosomes and plasmids
what is the function of nucleoid-associated proteins (NAPs) in bacteria?
- function in the cytoplasm rather than within a membrane-bound nucleus
- NAPS e.g. H-NS and IHF exert effects on prokaryotic gene expression
- synthesis of mRNA and protein are directly coupled in bacteria
what is the size and composition of bacterial ribosomes?
- 10-20 nm
- sedimentation coefficient of 70S: composed of a 30S and 50S subunit
what is the composition of eukaryotic ribosomes?
40S and 60S subunits in the 80S
what do multiple ribosomes form?
multiple ribosomes attach to single mRNA molecules to form polysomes
what are polysomes?
multiple ribosomes attached to single mRNA molecules
what is the thickness/structure of bacterial cytoplasmic membrane?
- 5-10 nm thick
- consists mainly of phospholipids and proteins
- contains integral, transmembrane, peripheral and anchored proteins
- protein rich, little space for phospholipids
what is the function of bacterial cytoplasmic membrane?
multifunctional nature
- protein secretion, packaging and processing
- electron transport and oxidative phosphorylation
what is the structure/characteristics of the cell wall?
- lies immediately external to the cytoplasmic membrane
- 10-25 nm thick
- strong, rigid (some elasticity), openly porous (permeable to solute molecules smaller than 10kDa in mass and 1 nm in diameter)
what is the function of the bacterial cell wall?
- supports the weak cytoplasmic membrane against the high internal osmotic pressure
- maintains the characteristic shape of the bacterium in its form
what are the internal osmotic pressures in Gram-positive and Gram-negative bacteria?
25 and 5 atm respectively
what is the main strengthening component of the bacterial cell wall?
peptidoglycan (syn. mucopeptide or murein)
what is peptidoglycan composed of?
N-acetylglucosamine and N-acetylmuramic acid molecules linked alternatively in a chain
what is the murein sacculus?
heteropolymer (peptidoglycan) forms a single molecular continuous sac external to the cytoplasmic membrane
what is involved in the Gram-stain procedure?
- distinguishes bacteria as Gram-positive or Gram-negative
- according to whether or not they resist decolouration with acetone, alcohol or aniline oil after staining with a triphenyl methane dye, e.g. crystal violet, and subsequent treatment with iodine
what are the results of the Gram stain procedure?
- Gram-positive bacteria resist decolouration and remain stained a dark purple colour
- Gram-negative bacteria are decolorized, and then counterstained light pink by the subsequent application of safaranin, neutral red or dilute carbol fuchsin
what peptides do the N-acetylmuramic acid units of peptidoglycan carry?
- L-alanine
- D-glutamic acid
- either meso-diaminopimelic acid (Gram-negative bacteria) or L-lysine (Gram-positive bacteria)
- D-alanyl-D-alanine
what is the strength of peptidoglycan walls given by?
cross-links that form between adjacent strands
- may be formed directly between meso-diaminopimelic acid or L-lysine of one strand and the penultimate D-alanine of the next, or through an interpeptide bridge composed of up to 5 amino acids
what can interfere with construction of cell wall peptidoglycan?
several antibiotics
what is lose in the cross-linking reaction in cell wall peptidoglycan?
terminal D-alanine
what do many Gram-positive bacteria contain in their cell wall?
- large amounts of teichoic acid (a polymer of ribitol or glycerol phosphate complexed with sugar residues) interspersed with the peptidoglycan
- some of this material (lipoteichoic acid) is linked to lipids buried in the cell membrane
what are the components of the envelope of the Gram-positive bacteria cell wall? what do they contain?
capsule: capsular polysaccharide
cell wall: peptidoglycan with teichoic acid polymers
cytoplasmic membrane: phospholipid bilayer with various membrane proteins, enzymes and permeases
what is the difference between teichoic acid and lipoteichoic acid in peptidoglycan cell wall in Gram-positive bacteria?
- teichoic acid is just in the peptidoglycan cell wall
- lipoteichoic acid is linked to lipids buried in the cell membrane
what is teichoic acid?
a polymer of ribitol or glycerol phosphate complexed with sugar residues
what is a structure of Gram-negative bacteria that Gram-positive bacteria don’t have?
- second outer membrane external to the peptidoglycan layer
- unit membrane in which the outer leaflet is largely composed of lipopolysaccharide (LPS)
what does the second outer membrane of Gram-negative bacteria contain?
- composed mainly of LPS
- contains associated proteins with functions including selective permeability (porins) and attachment (adhesins)
what properties does the outer membrane confer on Gram-negative bacteria?
- protects peptidoglycan from effects of lysozyme
- impedes the ingress of many antibiotics
what is N-acetylmuramic acid derived from?
N-acetylglucosamine by the addition of a lactic acid unit
how is peptidoglycan formed?
- each N-acetylmuramic acid molecule is substituted with a pentapeptide
- an N-acetylglucosamine molecule is joined to the muramylpentapeptide within the cell membrane
- unit is transferred to growth points in the existing peptidoglycan, where adjacent strands are cross-linked
what breaks the bond between N-acetylmuramic acid and N-acetylglucosamine?
lysozyme
what is contained within the outer membrane in Gram-negative bacteria?
- LPS
- porins
what is contained within the cell wall of Gram-negative bacteria?
- peptidoglycan
- lipoprotein
- periplasmic enzymes
what is contained within the inner membrane of Gram-negative bacteria?
- phospholipid bilayer with various membrane proteins, enzymes and permeases
what are the differences between the peptidoglycans of Gram-positive and Gram-negative organisms?
Gram-positive: cross-linking occurs through a peptide bridge (pentaglycine in S. aureus)
Gram-negative: direct cross linking
- terminal D-alanine is lost in both cases
what is the peptide structure of Gram organisms/peptidoglycans?
N-acetylmuramic acid linked to L-alanine linked to m-DAP to two D-alanine
what is involved in direct cross-linking?
m-DAP crosslinking
what do components of LPS form? what the core structure?
- core structure lipid A
- form endotoxin, which when released into the bloodstream, may give rise to endotoxic shock
what is the structure of mycobacteria?
- a third type of cell envelope is characteristic of mycobacteria
- causal agents of TB and leprosy
- peptidoglycan layer is covalently linked on its outer aspect to arabinogalactan, which is itself substituted with unique lipids (mycolic acids)
what are mycolic acids? where are they found?
- beta-hydroxy fatty acids consisting of 60 to 90 carbon residues
- together with non-covalently linked free lipids, form an extremely hydrophobic external layer which also contains porins
- found in mycobacteria
what property does mycobacteria’s envelope structure confer?
acid fast staining by methods such as Ziehl-Neelsen and phenol-auramine procedures
what is involved in the Ziehl-Neelsen method? what can it detect?
- detects the tubercle bacillus and other mycobacteria
- mycolic acids provide a barrier to simple aqueous stains
- when permeability is altered by heating or phenol, concentrated solutions of basic fuchsin and fluorescent dyes auramine and rhodamine can produce well-stained cells that resist decolorisation by strong acids in alcohol
how are decoloured non-acid-fast organisms counterstained?
in a contrasting colour with methylene blue or malachite green
how can modifications of the ZN method be useful?
for the demonstration of bacterial endospores and organisms such as Nocardia spp. and cysts of some protozoa, notably Cryptosporidium spp.
when are the continuous and relatively amorphus layers external to Gram envelopes called capsules, and when are they called microcapsules?
capsules: when the layer is fully hydrated and resolvable by light microscopy
microcapsule: when it is narrower, and detectable only by indirect, serological means or by electron microscopy
what does does the capsular gel consist of?
largely of water and has only a small content of solids
- in most species, the solid material is a complex polysaccharide, and in others its main constituent is polypeptide
what is loose slime?
an amorphous, viscid, colloidal material secreted extracellularly by some bacteria
what are the roles of capsules in bacteria?
- protect against phagocytosis, the lytic action of complement and bacteriophage invasion
- antibodies directed against capsular antigens can protect against infection
- protect cells against desiccation
- typing of bacteria
what does the production of extracellular polysaccharides provide?
a matrix within which biofilm formation can take place
what are the types of arrangements of flagella?
- peritrichous/lateral: originate from the sides of the cell
- polar: originate from one or both ends
what does the external portion of a flagellum consist of?
polymer of a single protein, flagellin
what does the basal region of the flagellum consist of?
multiple subunits that anchor and power the organ
how is the flagellar motor powered?
directly: by the protein gradient created across the cytoplasmic membrane by electron transport
how does Escherichia coli show chemotactic behaviour?
- alteration between the anti-clockwise and clockwise motion of the flagella effects, respectively, linear or tumbling motility
- intervals between these two patterns are modulated by chemical signals in the environment
what is the number/appearance of fimbriae?
100-500 per cell
0.1-1.5 μm in length
short
4-8 nm in width
how are fimbriae categorised? give examples of different types
- dimensions, antigenic and phenotypic properties
- types 1 and 2, P type
- present in both Gram-positive and Gram-negative bacteria
what are the roles of fimbriae?
important in mediating adhesion between the bacterium and host cells (recognised in the phenomenon of haemagglutination, a property of type 1, mannose sensitive pili)
what are sex pili? what are their roles?
- longer than fimbriae
- confer ability to attach specifically to other bacteria that lack these appendages
- initiate the process of conjugation
- act as receptor sites for certain bacteriophages described as being donor specific
what are the types of bacterial growth?
growing (vegetative), stationary or dead; uniform in their properties
at what levels does bacteria adapt to the environment?
phenotypic and genotypic levels
what is an endospore?
some bacteria, e.g. those of genera Bacillus and Clostridium, develop a highly resistant resting phase or endospore, whereby the organism can survive in a dormant state through long periods of starvation or adverse environmental conditions
what happens in sporulation and germination?
- each vegetative cell forms only one spore
- in subsequent germination each spore gives rise to a single vegetative cell
- doesn’t involve multiplication
what are sporulation stimuli?
classically starvation or transition from growth to stationary phase
what is the end result of sporulation?
a morphologically distinct structure, the endospore, within the mother cell
what are the appearances of spores?
- in unstained preparations, it has greater refractility
- larger than lipid inclusion granules and is often ovoid
- in phase-contrast microscopy, mature ungerminated spores are phase bright and immature germinated spores are phase dark
- when mature, spores resist colouration by simple stains
what are the types of spore appearances?
varies according to the species
- spherical, ovoid or elongated
- occupying a terminal, subterminal or central position
- being narrower than the cell, broader or bulging it
what is an exosporium?
additional, apparently loose covering of spores
why are spores more resistant than vegetative cells?
- high content of calcium and dipicolinic acid
- low content of water
- very low metabolic and enzymic activity
what is reactivation of a spore called?
germination
when does germination of a spore occur? what does it involve?
- occurs in response to specific stimuli that are related to external conditions favourable to growth
- irreversible and involved rapid degradative changes
- spore loses its heat resistance and its dipicolinic acid; loses calcium; becomes permeable to dyes and its refractivity changes
what happens to a spore during germination?
spore swells, its coat is broken open and a single vegetative cell emerges
is the state of dormancy of spores alterable?
yes
what is outgrowth?
after germination, cell growth leading up to the formation of the first vegetative cell and before the first cell division is outgrowth
what is the structure/cross-section of a bacterial spore?
- spore cytoplasm (core)
- inner membrane surrounds the core
- spore cortex
- spore coat (resistant/multiple layers)
- exosporium (loose outer covering)
what are conidia (exospores)? what forms them? what are they vulnerable/resistant to?
- some mycelial bacteria (Actinomycetales) and many filamentous fungi form conidia
- resting spores that are different from endospores
- borne externally by abstriction from the ends of the parent cells (conidiophores)
- are disseminated by the air or other fresh habitats
- not resistant to heat and disinfectants
what is pleomorphism of bacteria?
- bacteria of a single strain may show variation in size and shape, or form a proportion of cells that are swollen, spherical, elongated or pear shaped
where does pleomorphism occur most readily?
- in certain species (e.g. Streptobacillus moniliformis and Yersinia pestis) in ageing cultures
- species on artificial medium
- species in presence of antagonistic substances eg. penicillin, glycine, lithium chloride, sodium chloride in high concentrations and organic acids at low pH
what are involution forms? what is their abnormal shape due to?
abnormal bacterial cells formed by pleomorphism
- some are non-viable, whereas others may grow and revert to the normal form when transferred to a suitable environment
- abnormal shape is due to defective cell wall synthesis and produces a swollen cell, which bursts and lyses
how can bacteria be converted into viable spherical bodies?
if bacteria have their cell walls removed or weakened while they’re held in a solution of sufficient osmolarity to prevent them taking up water by osmosis, they may escape being lysed and become converted
what are free protoplasts?
bacteria that have been converted into viable spherical bodies which have had all cell wall material removed from them
what are spheroplasts?
bacteria that have been converted into viable spherical bodies which remain enclosed by an intact, but weakened residual cell wall
how can protoplasts and spheroplasts vary in size?
- osmotically sensitive
- vary in size with the osmotic pressure of the suspending medium
- if medium is much diluted, they swell up and perish by lysis
what are L-forms of bacteria?
- may arise spontaneously
- cell wall deficient
- difficult to demonstrate as they don’t stain with Gram or acid-fast methods and may not propagate in vitro
what are the types of groups that microorganisms can be classified into?
- algae
- protozoa
- slime moulds
- fungi
- bacteria
- archaea
- viruses
what types of microorganisms are prokaryotic?
- algae (excluding blue-green algae)
- protozoa
- slime moulds
- fungi
what types of microorganisms are eukaryotic?
- bacteria (including organisms of the mycoplasma, rickettsia and chlamydia groups)
- blue-green algae
what are viroids?
protein-free fragments of single-stranded circular RNA that cause disease in plants
what are prions?
- naturally occurring host cell membrane glycoproteins that undergo conformative changes to an infectious isoform
- causative agents of fatal neurodegenerative disorders
what is taxonomy?
- classification, nomenclature and identification of microbes)
- consists of three components: classification, nomenclature and identification
what is systematics?
- concerned with evolution, genetics and speciation of organisms
- referred to as phylogenetics
how are protozoa, fungi and helminths classified and named?
- according to work from Linnaeus
- genus, then species, then variant
what tests of bacteria are definitive of a genus or species?
universal production of catalase enzyme and cytochrome c, respectively, by Staphylococcus spp. and Pseudomonas aeruginosa
what are the taxonomic ranks used in classification of bacteria?
Kingdom (Prokaryotae) Division (Gracilicutes) Class (Betaproteobacteria) Order (Burkholderiales) Family (Burkholderiaceae) Genus (Burkholderia) Species (Burkholderia cepacia)
what are the different methods of classification of bacteria?
- Adansonian or numerical classification
- DNA composition
- DNA homology
- ribosomal RNA sequencing
what are subcategories of eukaryotes that are of medical importance?
protozoa and fungi
what are examples of protozoa?
sporozoa: plasmodium, isospora, toxoplasma, cryptosporidium
flagellates: giardia, trichomonas, trypanosoma, leishmania
amoebae: entamoeba, naegleria, acanthamoeba
other: babesia, balantidium
what are examples of fungi?
mould-like: epiderophyton, trichophyton, microsporum, aspergillus
yeast-like: candida
dimorphic: histoplasma, blastomyces, coccidioides
true yeast: cryptococcus
what are examples of actinobacteria?
(High G+C Gram positives)
actinomyces, streptomyces, corynebacterium, nocardia, mycobacterium, micrococcus
what are examples of firmicutes?
gram-positive bacilli: listeria, bacillus, clostridium, lactobacillus, eubacterium
gram-positive cocci: staphylococcus, streptococcus, enterococcus
gram-negative cocci: veillonella, mycoplasma
what are examples of proteobacteria?
gram-negative cocci: neisseria, moraxella
gram-negative bacilli: enterobacteria - escherichia, klebsiella, proteus, salmonalla, shigella, yersinia
pseudomonads: pseudomonas, burkholderia, stenotrophomonas, haemophilus, bordetella, brucella, pasteurella
gram-negative curved and spiral bacilli: vibrio, spirillum, campylobacter, helicobacter
what are examples of bacteroidetetes?
bacteroides, prevotella
what are examples of spirochaetes?
borrelia, treponema, brachyspira, leptospira
what are actinobacteria?
- gram-positive bacteria with a high G + C content
- many capable of filamentous growth with true branching which may produce a mycelium
- many do not stain well with the Gram method
- includes mycobacteria which can be recognised with acid fast stains
what are firmicutes?
- low G + C Gram-positive bacteria including bacilli
- includes most of the medically significant Gram-positives
- Veillonella and Mycoplasma, which have presumably lost their Gram-positivity, are included
what are proteobacteria?
- very large group of Gram-negative bacteria (bacilli and cocci)
- five subdivisions: alpha, beta, gamma, delta and epsilon
- rods, most are motile
- facutatively anaerobic
- some colonise the GI tract
- enterobacteriaceae
what are bacteroidetes?
Gram-negative anaerobes
what are spirochaetes?
possessing cells with a tight spiral shape and an internal flagellum
what are chlamydiae?
strict intracellular parasites
what are some categories of actinobacteria?
- actinomyces
- streptomyces
- mycobacterium
- nocardia
- corynebacterium
what are features of actinomyces? give examples
gram-positive, non-acid-fast, tend to fragment into short coccal and bacillary forms and not to form conidia; anaerobic
- e.g. Actinomyces israelii
what are features of streptomyces? give an example
- vegetative mycelium does not fragment into short forms
- conidia form in chains from aerial hyphae
- e.g. Streptomyces griseus
what are features of Mycobacterium? give an example
- acid-fast
- Gram-positive, but does not readily stain by the Gram method
- usually bacillary, rarely branching, make mycolic acids
- e.g. Mycobacterium tuberculosis
what are features of Nocardia? give an example
- similar to Actinomyces, but aerobic and mostly acid-fast
- make mycolic acids
- e.g. Nocardia asteroides
what are features of Corynebacterium? give an example
- pleiomorphic
- Gram-positive bacilli
- make mycolic acids
- e.g. Corynebacterium diphtheriae
what are types of Firmicutes?
- Streptococcus
- Staphylococcus
- Mycoplasma and ureoplasma
- Veillonella
- Gram-positive spore-forming bacilli
- Gram-positive non-sporing bacilli
what are features of Streptococcus? give an example
- Gram-positive cocci
- mainly adherent in chains due to successive cell divisions occuring in the same axis (e.g. Streptococcus pyogenes)
- sometimes predominantly diplococcal (e.g. Streptococcus pneumoniae)
what are features of Staphylococcus? give an example
- Gram-positive cocci
- mainly adherent in irregular clusters due to successive divisions occurring irregularly in different planes (e.g. Staphylococcus aureus)
what does the ZN stain detect?
Mycobacterium/acid-fast bacilli
what is LPS/endotoxin made up of?
terminal sugars, O antigen and Lipid A
what is between the outer and inner membrane in Gram-negative bacteria?
- lipoprotein
- periplasmic space
- some peptidoglycan
what is the goal of spore formation? what do they contain?
- certain harsh conditions; spores are resistant to them
- contain DNA
- wait for better conditions
- resist desiccation
what is bacterial environment?
- less than -80 to +80 degrees (120 for spores)
- pH less than 4 to 9
- water/dessication: 2hours to 3 months (>50 years for spores)
- UV light
what are the growth conditions and doubling time for viruses, E.coli/S.aureus etc, Mycobacterium tuberculosis, Fungi, Mycobacterium leprae
most viruses: cells; < 1 hour
E.coli, S.aureus etc: broth or solid media; 20-30 mins
Mycobacterium TB: broth or media; 24 hours
Fungi: broth or media; 30 minutes
Mycobacterium leprae: broth or media; 2 weeks
what is endotoxin?
component of the outer membrane of bacteria, e.g. LPS in Gram-negative bacteria
what is exotoxin?
secreted proteins of Gram-positive and Gram-negative bacteria
what is the composition, action, effect of heat, antigenicity, production by, and convertibility to toxoids of exotoxins vs endotoxins?
exotoxins:
- protein
- specific action
- labile (heat effect)
- strong antigenicity
- produced by mostly Gram-positive bacteria
- can convert to toxoid
endotoxins:
- LPS
- non-specific action
- stable (heat effect)
- weak antigenicity
- produced by Gram-negative bacteria
- not convertible to toxoid
how does gene transfer occur in bacteria?
transformation e.g. via plasmid
transduction e.g. via phage
conjugation e.g. via sex pili
what are obligate intracellular bacteria? what are examples of each?
Rickettsia
- R. rickettsii
- R. prowazekii
- R. conorii
Chlamydria
- C. trachomatis
- C. psittaci
- C. pneumoniae
Coxiella
- C. burnetii
what are examples of bacteria that can be cultured on artificial media without a cell wall?
Mollicutes
- Mycoplasma pneumoniae
- M. hominis
- Ureaplasma urealyticum
what are categories of bacteria that can be cultured on artificial media with a cell wall?
- growing as single cells
- growing as filaments
what are types of bacteria with a cell wall that grow as filaments? give examples of each
Actinomyces
- A. asraelii
Nocardia
- N. asteroides
Streptomyces
what are categories of bacteria growing as single cells that have a cell wall?
- rods
- cocci
- spirochaetes
what are types of spirochaetes? give examples of each
Leptospira
- L. icterohaemorrhagiae
Treponema
- T. pallidum
Borrelia
- B. burghdorferi
- B. recurrentis
what is the mechanism of action of the Gram stain on Gram-negative bacteria?
decolouriser interacts with the lipids and cells lose their outer LPS membrane and the crystal violet-iodide complexes; appear pink with counterstain
what is the mechanism of action of the Gram stain on Gram-positive bacteria?
the decoloriser dehydrates the cell wall and the CV-I complex gets trapped in the multi-layered peptidoglycan
what in the Gram stain is used as a decoloriser?
ethanol or acetone
what are categories of cocci?
Gram-negative and Gram-positive
what are categories of Gram-negative cocci? what are examples of each?
anaerobic: Veillonella
aerobic: Neisseria
- N. meningitidis
- N. gonorrhoeae
what are categories of Gram-positive cocci?
aerobic and anerobic
what is a type of anaerobic Gram-positive cocci?
Peptostreptococcus
what are types of aerobic Gram-positive cocci? give examples for each
Staphylococcus
- S. aureus
- S. epidermidis
Streptococcus
what are types of Streptococcus? give examples for each
Beta-haemolytic
- S. pyogenes (A)
- S. agalactiae (B)
Alpha-haemolytic
- S. pneumoniae
- S. oralis
- S. milleri
- S. sanguis
Non-haemolytic
- S. bovis
Enterococcus
- E. faecalis (D)
what is involved in the catalase test?
- flavoproteins reduce O2 using superoxide dismutase producing H2O2
- superoxide is converted by catalase to H2O and O2
what bacteria are catalase+? which aren’t?
- staphylococci are catalase +
- many Gram-negative bacteria and fungi are also catalase +
- streptococci are not catalase +
what is the goal of the coagulase test?
- distinguishes S. aureus (coagulase positive) from other staphylococci (coagulase negative)
what does coagulase do? how is it detected in the coagulase test?
coagulase activates prothrombin to convert fibrinogen to fibrin
- free coagulase is released and identified by tube coagulase test
- bound coagulase (clumping factor) is identified by a slide test
- negative remains liquid, positive clumps
what are examples of coagulase positive and negative staphylococci?
coagulase +: S. aureus
coagulase -: S. epidermidis
what is haemolysis? what does it require? what does it classify?
- the ability of bacteria to break down erythrocytes in blood agar
- requires expression of hemolysin
- useful for classifying streptococci
what is alpha haemolysis? what does it look like?
- caused by hydrogen peroxide produced by the bacterium, oxidising hemoglobin and producing the green oxidised derivative methemoglobin
- green/incomplete hemolysis
- agar under the colony is dark and greenish
what are examples of bacteria that display alpha haemolysis?
Streptococcus pneumoniae
Streptococcus viridans
what is beta haemolysis? what does it look like? what is it caused by?
- complete haemolysis
- complete lysis of red cells in media around and under colonies
- lightened (yellow) and transparent
- streptolysin, an exotoxin, is the enzyme produced by bacteria that causes the complete lysis of erythrocytes
what are types of streptolysins in beta haemolysis?
streprolysin O
- oxygen sensitive cytotoxin secreted by most group A streptococcus and streptococcus dysgalactae
- interacts with cholesterol in membrane of eukaryotic cells
streptolysin S
- oxygen-stable cytotoxin produced by most GAS strains leading to clearing on surface of blood agar
what is gamma haemolysis?
- non-haemolytic
- organism doesn’t induce haemolysis
- agar under and around the colony is uncharged
what are examples of organisms displaying gamma haemolysis?
- enterococcus faecalis
- Staphylococcus saprophyticus
- Staphylococcus epidermidis
what organisms produce beta-haemolysis?
S. pyogenes
what are examples of mycobacteria?
M. tuberculosis M. leprae M. avium-intracellulare M. ulcerans M. kansasii
what are categories of Gram-positive rods?
aerobic and anaerobic
what are types of anaerobic Gram-positive rods? give examples for each
Clostridium
- C. perfringens
- C. tetani
- C. botulinum
- C. difficile
Proprionibacterium
- P. acnes
what are types of aerobic Gram-positive rods? give examples for each
Corynebacterium
- C. diphtheriae
Listeria
- L. monocytogenes
Bacillus
- B. anthracis
- B. cereus
Erysipelothrix
- E. rhusiopathiae
what are features of MacConkey agar?
can be used for Gram-negative bacilli
- bile salts inhibit Gram-positive bacteria and inhibit swarming of Gram-negative bacterium Proteus spp.
- crystal violet inhibits some Gram-positive bacteria
- neutral red acts as a pH indicator so bacteria that ferment lactose and release H+ appear pink or red
what is CLED media? what is it used for?
Cysteine lactose electrolyte deficient media
- prevents swarming of Proteus mirbilis
- Bromothymol blue is indicator, lactose fermentation causes blue to yellow change, decarboxylation of L-cyteine results in blue colour
- used as media for urinary pathogens
what are lactose fermentors (gram-negative aerobes)?
E. coli
Klebsiella pneumoniae
Enterobacter spp.
what are slow fermentors (Gram-negative aerobes)?
Serratia spp.
Citrobacter spp.
what are lactose non-fermentors (Gram-negative aerobes)?
Salmonella spp. Shigella spp. Yesrinia spp. Proteus spp. Pseudomonas aeruginosa
what is the oxidase test used for?
- tests if micro-organism contains a cytochrome oxidase or indophenol oxidase
- utilises a redox indicator e.g. N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD)
- in oxidised state: dark blue/ maroon
- implies organism is able to use oxygen as the terminal electron acceptor
what are examples of oxidase+ microorganisms?
pseudomonads, aeromonads, curved rods and Neisseria spp.
what does LPS consist of?
- lipid A, the toxic portion of LPS that is anchored in the outer leaflet of the outer membane
- core (R) antigen (core-oligosaccharide), short chain of sugars, some are unique to LPS
- somatic (O) antigen (O polysaccharide), a highly antigenic repeating chain of oligosaccharides
why do mycobacteria not stain as Gram-positive?
- mycobacteria have an outer lipid bilayer composed of mycolic acids
- mycoplasmas lack peptidoglycan
what are pathogenicity determinants/virulence factors?
any product or strategy that contributes to pathogenicity/virulence
what are categories of pathogenicity determinants?
colonisation factors: adhesins, invasins, nutrient acquisition, defence against the host
toxins: usually secretetd proteins (by protein secretion systems) leading to damage and subversion
what is the lactose use, lifestyle and motility of Shigella flexneri? (enterobacteria)
- no lactose use
- primate pathogen
- not motile
what is the lactose use, lifestyle and motility of Escherichia coli? (enterobacteria)
- does use lactose
- mammalian commensal and pathogen
- is motile
what is the lactose use, lifestyle and motility of Salmonella enterica? (enterobacteria)
- no lactose use
- mammalian pathogen
- is motile
what are cell surface antigens of Gram-negative bacteria?
H antigen (flagella) O (somatic) antigen (LPS K antigen (exopolysaccharide capsule)
what are principal infections caused by pathogenic Escherichia coli strains?
- wound infections (surgical)
- UTIs
- gastroenteritis
- travellers’ diarrhoea
- bacteraemia (sometimes leading to sepsis)
- meningitis
what is the mechanism of ETEC heat labile enterotoxin?
- stimulates heat labile enterotoxin
- stimulates Gs, activates adenylate cyclase, increases cAMP, activates PKA and activates the CFTR
- activates CFTR; transport of Cl-/H2O out of cell at apical surface
what is the mechanism of ETEC heat stable enterotoxins?
- activation of guanylate cyclase
- increased cGMP, activation of cGMP-dependent protein kinase, which activates CFTR
what is ETEC, PEC, and EHEC?
enterotoxigenic
enteropathogenic
enterohaemorrhagic
what is EIEC, EAEC and UPEC?
enteroinvasive
enteroaggregative
uropathogenic
why are some E. coli strains pathogenic?
- several pathovars (distinct pathogenic strategies)
- common core genome
- acquisition of pathogenicity genes (lateral gene transfer)
what are the effects of EHEC/EPEC induced pedestal formation?
diarrhoea symptoms caused by disruption of tight junctions and effects on ion secretion
what is the process of EHEC/EPEC-induced pedestal formation?
- initial adherence by bundle-forming pili
- T3SS (syringe-like protein secretion system) activity - effector secretion. Actin microfilament depolymerisation, microvilli effacement
- injection of Tir (translocated intimin receptor) - intimin-dependent signalling
- actin nucleation, pedestal formation, intimate attachement
- intimin is a bacterial adherence protein
what is the pathogenesis of GI infection caused by EPEC in the SI?
destruction of microvilli and entrance into cells
what is the pathogenesis of GI infection caused by ETEC in SI?
heat labile enterotoxin and heat stabile enterotoxin
what is the pathogenesis of infection caused by EHEC, EIEC and EAEC in the colon?
shiga-like toxin
heat stable enterotoxin like haemolysin
what are the different species of shigella?
S. dysenteriae
S. flexneri
S. boydii
S. sonnei
what does Shigella cause?
- severe bloody diarrhoea
- frequent passage of stools (>30/day)
- small volume, pus and blood, prostrating cramps, pain in straining, fever
- self limiting in adults
what is the pathogenesis of Shigella infection? how is it spread?
- acid-tolerant
- person-to-person, contaminated water and food
- entry through colonic M cells (antigen sampling, overlie lymphoid follicles)
- induced uptake
what happens in invasion of the colonic mucosa by Shigella?
- entry through M cells on enterocyte apical surface
- shigella passes through tight junctions and spread through the cells
- apoptotic macrophages
- translocating PMNs to the site of infection
what is the shiga toxin?
- catalytic subunit (glycosidase)
- receptor binding subunits
what is the action of the Shiga toxin?
- cleaves N-glycosidic bond of adenosine residue in 28S rRNA (60S subunit)
- blocks EF-1 and EF-2 binding -> protein synthesis inhibited
what complication does the Shiga toxin cause?
systemic absorption of Shiga toxin -> targets kidney -> HUS (microvascular thrombosis in kidneys -> kidney failure
what are the main clinically relevant Gram-negative bacteria?
- Escherichia coli
- Shigella
- Salmonella
what are the species of Salmonella?
S. enterica - responsible for salmonellosis
S. bongori - rare (contact with reptiles)
what are the three salmonellosis caused by S. enterica?
- gastroenteritis/entercolitis (serovars Enteritidis and Typhimurium)
- Enteric fever - typhoid (serovars Typhi and Paratyphi)
- Bacteraemia (serovars Cholerasuis and Dublin)
what is the pathogenesis of salmonellosis?
- invasion of gut epithelium (SI)
- intestinal secretory and inflammatory response (serovars Enteritidis and Typhimurium)
- transcytosed to basolateral membrane
- enters submucasal Ms
- intracellular survival/replication
how is salmonella spread?
ingestion of contaminated food/water
how does salmonella lead to systemic infection?
dissemination within Ms (serovar Typhi)
what are the steps in pathogenesis of gastroenteritis caused by salmonellosis?
- bacterial-mediated endocytosis
- induction of chemokine release
- neutrophil recruitment and migration
- neutrophil-induced tissue injury
- fluid and electrolyte loss -> diarrhoea
- > inflammation/necrosis of the gut
- no systemic spread
what is the pathogenesis of enteric fever caused salmonellosis
- bacterial-mediated endocytosis
- transcytosis to basolateral membrane
- survival in M -> systemic spread
- > initially, little damage to gut mucosa
- migration to reticuloendothelial organs via lymphatics and blood
what are the effects of typhoid (enteric) fever?
ingestion of S. Typhi -> small intestine -> inflammation and ulcertation of Peyer’s patches -> diarrhoea: haemorrhage or perforation (1-3% of cases)
multiplication in macrophages -> secondary bacteraemia (septicaemia) -> fever: kidney and other organs affected
gall bladder -> carrier state (1 year to rest of life)
how is typhoid (enteric) fever dealt with by the body?
ingestion -> small intestine -> mesenteric lymph nodes -> transient bacteraemia (thoracic duct - enter bloodstream) -> multiplication in macrophages in liver, spleen and bone marrow -> gall bladder (from liver) -> small intestine (via bile)
what is the incubation period of typoid (enteric) fever?
1-3 weeks; asymptomatic
what is Proteus mirabilis?
differentiates into an elongated hyperflagellated form known as a swarmer cell -> surface motility
- normally flagellated form is the swimmer cell -> motility in liquid
what can Proteus mirabilis lead to? what is its virulence factor?
- opportunistic
- UTI -> pyelonephritis, septicaemia
- virulence factor: urease (increased pH) -> kidney stones
what is Klebsiella pneumoniae? what does it do?
- environmental
- opportunistic, nosocomial infections (neonates, elderly, compromised)
- colonisation of GIT (normal) and oropharynx (less frequent) is benign
- can lead to UT, pneumonia, surgical wound infections, bacteraemia - sepsis
- MDR (resistant to carbapenems)
what is Enterobacter (E. cloacae, E. aerogenes)? what does it lead to?
- opportunic, nosocomial outbreaks
- infections in lungs, urinary tract, abdominal cavity, IV devises, sepsis
- spread from endogenous gut flora, can survive on skin, patient-to-patient transmission
- cephalosporin resistant forms
what is Yersinia spp.? what does it lead to?
- primary pathogen
- Y. enterocolitica: localised to ileum; gastroenteritis
- Y. pestis: systemic; bubonic plague
what is Vibrio cholerae? what does it lead to?
- facultative anaerobe
- curved rods with a single polar flagellum
- saline environments: commensal to planktonic crustaceans e.g. copepods
- ingestion by shellfish, contamination of drinking water due to flooding of coastal areas or poor sanitation
what is the incubation time for Vibrio cholerae?
hours to 5 days (multiplies in small intestine)
what are the virulence determinants for Vibrio cholerae?
- TCP pili: required for colonisation
- cholera toxin (A and B subunits)
what is the action of cholera toxin?
- binds a ganglioside (glycolipid) receptor (B subunits)
- A subunit ADP-ribosylates Gs -> locked in ON state
- uncontrolled cAMP production
- protein kinases activated
- CFTR ion transporter activity modified (loss of Cl- and Na+)
- massive loss of H2O
what is Pseudomonas aeruginosa? what does it lead to?
- ubiquitous, free-living, aerobe
- motile: single polar flagellum
- opportunistic (serious cause of nosocomial infections)
- multiple antibiotic resistance
- very difficult to treat
what are types of acute infections caused by P. aeruginosa?
localised
- burns/surgical wounds
- UTI (catheters)
- keratitis
systemic (bacteraemic -> sepsis)
- neutropenic patients (leukaemia, chemotherapy, AIDS)
ICU patients (ventilators) - leading cause of nosocomial pneumonia
what is a type of chronic infection caused by P. aeuginosa?
CF
what are P. aeruginosa virulence determinants?
multiple toxins: main virulence determinants in acute infections
- exoenzyme S and U interfer with cell signalling
- exotoxin A, elastase, phospholipase cause cell death/damage
- cyanide
CF: copious amounts of exopolysaccharide alginate is a virulence factor
what is Haemophilus influenzae? how is it carried?
- exclusively human parasite
- nasopharyngeal carriage in 25-80% of the population (non-capsulate)
- transient carriage of capsulate strains in 5-10%
what can Haemophilus influenzae lead to?
opportunistic infections seen mainly in young children and adult smokers
- meningitis (< age 5 years), 5-10% of adult cases
- bronchopneumonia
- epiglottitis, sinusitis, otitis media
- bacteraemia (often associated with pharyngitis)
- pneumonia in CF, COPD, HIV patients
what is chocolate agar?
blood agar which has been heated to 80 degrees to allow release of haem and NAD by erythrocytes. colour of the agar is brown rather than red
what are virulence determinants of H. influenzae?
capsule - invasive strains are encapsulated
- can penetrate nasopharyngeal epithelium
- resistance to phagocytosis and complement system
- capsule allows typing into 6 serogroups (a-f)
LPS
- inflammation
- complement resistance
what is NTHi?
non-typeable H. influenzae
- commensals and upper respiratory tract pathogens are non-capsulate
- capsule allows typing into 6 serogroups (a-f)
- type b strains are main cause of meningitis
what is Legionella pneumophilia? what does it cause?
- discovered 1976
- Lesionnaires’ disease -> severe inflammatory pneumonia
- affects the immunocompromised
- severe (15-20% mortality)
how does Legionella pneumophilia grow?
- fastidious: culture on charcoal agar
- man-made aquatic environments
- replicate within freshwater protozoa - intracellular parasite of amoeba
- can infect alveolar macrophages
what is Bordetella pertussis? what does it cause?
- pertussis (whooping cough)
- B. parapertussis causes mild pharyngitis
- short (sometimes oval) rods (coccobacilli)
- fastidios, non-invasive, highly contagious (aerosol transmission)
- obligate human pathogen
what is the mechanism of action of B. pertussis?
- predilection for ciliated epithelia
- binds to respiratory epithelial cells
what are the toxins in B. pertussis? how do they act?
Pertussis toxin
- S1 subunit ADP-ribosylates Gi -> locked in OFF state
- PT -> increased cAMP
adenylate cyclase-haemolysin toxin (CyaA)
- hypersynthesis of cAMP -> suppression of innate immune functions
what is Neisseria? where is it present?
- non-flagellated diplococci
- fastidious
- obligate human pathogen
- present in PMNs of CSF (N.m) or urethral discharge (N.g.) during infection
what are the two medically important forms of Neisseria?
N. meningitidis
N. gonorrhoeae
what is the incidence of N. meningitidis? how does it spread?
- nasopharynx of 5-10% of the population (asymptomatic)
- 20-90% during outbreaks
- person-to-person (aerosol) transmission (universities, barracks, Haj)
what is the pathogenesis of N. meningitidis?
- crosses nasopharyngeal epithelium and enters bloodstream in small proportion of colonised individuals
- > low level bacteraemia (asymptomatic) or septicaemia (sepsis)
- > meningitis: invasion of the meninges; bacteria enter CSF of subarachnoid space from blood stream after crossing BBB
- very high mortality of septicaemic form if not treated
what are the virulence determinants of N. meningitidis?
capsule (serogroup B in 90% of cases)
-> antiphagocytic
noncapsulated N.m. only found in nasopharynx - not pathogenic
LPS (membrane blebs)
- > cytokine cascade
- > sepsis
what are fungi? what are their characteristics?
- large, diverse group of heterotrophic organisms
- mostly found as saprobes in the soil and on decomposing organic matter
- eukaryotic
- have a range of internal membrane systems, membrane bound organelles, and a well defined cell wall
what is the fungal cell wall made up of?
composed largely of polysaccharides (glucan, mannan) and chitin
what are the three main groups that fungi can be divided into?
- moulds (multicellular filamentous fungi)
- yeasts
- dimorphic fungi
what are moulds composed of? how do they reproduce? do they produce spores?
- multicellular filamentous fungi
- composed of branching filaments, termed hypae, that grow by apical extension ot form an intertwined mass (mycelium)
- hyphae often have regular cross walls
- reproduce by asexual spore production (mitosis) or by sexual reproduction (meiosis)
what are hyphae (in fungi)?
branching filaments that grow by apical extension to form an intertwined mass (mycelium)
what is a mycelium (in fungi)?
an intertwined mass formed by growth of hyphae by apical extension
what are yeasts? how do they reproduce?
- predominantly unicellular and oval or round in shape
- most propagate by an asexual process called budding where the cell develops a protuberance, which enlarges and eventually separates from the parent cell
- some produce chains of elongated cells (pseudohyphae)
- some reproduce by fission
what is budding (in fungi)?
asexual process used by yeasts where the cell develops a protuberance, which enlarges and eventually separates from the parent cell
what are pseudohyphae (in fungi)?
- produced by yeasts
- chains of elongated cells that resemble the hyphae of moulds
what are dimorphic fungi?
group of fungi that are capable of changing their growth to either a mycelial or yeast phase, depending on growth conditions
