Environmental Interactions Flashcards
What are elements affecting survival of pathogens?
Starts at introduction
e.g Soil
= moisture
= surface / injected
= high / low organic matter
= predators
= dry / wet
= frozen / cold / warm
= plants / bare
= pH
e.g. Water
= ground / surface
= predators
= mixed / stagnate
= high / low nutrients
= high / low sediments
= frozen / cold / warm
= pH
What are Amoeba resistant bacteria ?
= enhanced survival
= some microorganisms have evolved to become resistant to protists and exit free-living amoebae after internalisation undamaged
= some are pathogens
What is the bacterial association with protozoa?
Amoebae
= diverse group within protozoa
= ubiquitous (soil, water, biofilms)
= abundance and diversity (season, temperature, moisture, precipitation, pH, nutrient availability)
= feed mainly on bacteria, fungi , algae by phagocytosis
= digestion occurs within phagolysosome
= 20% of clinical and environmental Acanthamoebae (host) harbour bacteria
= some amoebae are pathogenic (along with bacteria they contain)
= contain diverse range of bacteria
(α-, β-, γ- ε- proteobacteria + chlamydiae + flavobacteria + bacilli + actinobacteria)
= e.g. Chlamydia pneumoniae - Acanthamoeba spp.
e.g. Legionella pneumophila - many spp of amoebae
e.g. E. coli O157:H7 - Acanthamoeba spp.
e.g. Mycobacterium avium subspecies paratuberculosis (MAP) - Acanthamoeba spp.
What are the two stage in the amoeba life cycle?
Trophozoite
= metabolically active stage
= feeds on bacteria
= multiplies by binary fission
Cysts
= generally two layers (ectocyst, endocyst)
= some species have 3rd layer (mesocyst)
= provides resistance to adverse conditions
(e.g. disinfection, diver pH, osmotic pressure, temperature, nutritional state)
= exit when environmental conditions become favourable
What is the Legionella spp?
Legionella pneumophila
= flagellated γ-proteobacteria, acid fast, pleomorphic, non-sporulating, gram-negative rod (coccobacillus)
= Legionnaire’s disease OR legionellosis
= has produced many outbreaks
e.g. Philadelphia, Barrow in Furness
=also Legionella …
longbeachae
micdadei
anisa
hackeliae
dumoffi
gratiana
(57 species)
What does Legionella pneumophila cause?
= transmitted by inhalation of infected aerosols
Pontiac Fever
= self limited febrile respiratory illness in healthy people
= flu like, no pneuomina, rarely fatal
Legionnaire’s Disease
=severe pneuomina with systemic complications in older and immunocompromised
Populations at risk
= elderly , smokers, immunocompromised, men
Mortality rate
= 10-20%
= up to 50% in nosocomial infections
What is the ecology of Legionella pneumophila?
Aquatic reservoir
(main source: transmission)
= natural environments: lakes, rivers
= artificial environments: showers, taps, AC systems, cooling towers
Resistance to high temperatures
= warm water up to 50oC
Extracellular and Intracellular bacterium
= ecological niche: free living or within free living amoebae
Why is Legionella Pneumophila classed as a new pathogen?
Ubiquitous in environment BUT never previously described
= stain poorly for visualisation
= nutritionally demanding (not isolated on common microbiological media - needs iron salts, cysteine)
= first cultured in 1977
Originally found in ‘unsuitable’ environment
= unusual (AC units)
Found to invade protozoa as well as humans
Survives as intracellular parasites
What is the Epidemiology of Legionella sp?
24 species isolated from humans
BUT legionella pneumophila = 91% of cases worldwide
Legionella longbeachae
= 5% of cases worldwide
= 30% of cases in Aus/Nz
Legionella micdadei
= 2% cases worldwide
15 serogroups (sg) within Legionella pneumophila
Legionella pneumophila sg1:
= 88.6% of leigionellosis caused by L. pneumophila sg1
= seems to be more virulent to humans
What are the symptoms of a L. pneumophila infection?
Fever, chills
Non-productive cough
Headache
Pneumonia
Complications:
= GI, CNS, liver, kidneys
What is the life history of L. pneumophila?
What are the major virulence factors in Legionella spp?
Adherence
Endotoxin
Enzyme
Iron Uptake
Motility
Nutrient acquisition
Regulation
Secretion system
Stress protein
Toxin
Unclassified
How does L. pneumophila interact with macrophages, monocytes and amoebae?
= manipulates host cell vesicular-trafficking pathways and establishes a membrane-bound replication vacuole (MRV)
Intracellular lifestyle allows them to gain the following competitive advantages over other microorganisms
= evasion of predators in the environment
= evasion of host’s humoral and cellular immune responses
= easy access to all necessary nutrients
Must solve these problems:
= prevent newly formed vacuole from merging into antimicrobial lysosomal network
= manage nutrients acquisition through vacuolar membrane
= deal with space limitations during proliferative stage
Dual host system
= allows intracellular growth in protozoa and in human macrophages
Protozoa essential for Legionella growth
= interaction with amoeba generates pool of virulence traits during evolution
Intracellular pathogen invades and replicates within a protective phagosome inside alveolar macrophages
What are the 2 phases of growth of L. pneumophila?
Replicative Phase
= sodium resistance
= non-flagellated
= low-cytotoxicity
Infectious Phase
= short, thick
= flagellated
= high-cytotoxicity
Key to virulence of L. pneumophila is ability to prevent phagosome-lysosome fusion
What is the life cycle of L. pneumophila?
- Free-swimming transmissive phase
= L. pneumophila engulfed by phagocytic cells (amoebae / alveolar macrophages)
= establish vacuoles that provide protection from lysosomal digestion - If conditions favourable, intracellular bacteria repress transmission traits and activate pathways that promote replication phase
- Conditions deteriorate, progeny stop dividing, express traits to promote survival in environment and transmission to new phagocytic host
- After prolonged perioid, microbes continue to develop into mature intracellular form (MIF) - high resilient and infectious
- Phagocyte host lysed, microbes released into aqueous environment
- L. pneumophila that do NOT immediately encounter new phagocyte = establish biofilms in water systems (resistant to biocidal agents)
- Planktonic microbes encounter new phagocyte = cycle begin again
- Microbes cultured in broth display many of same traits of the replicative (if exponential phase) or transmissable (if in stationary phase) forms seen in phagocytes
How is the replication vacuole formed?
After uptake into macrophage, LCV evades transport to lysosomal network (sequestered)
Vesicles derived from ER + mitochondria appear closely
Vesicles that surround LCV (legionella containing vacuole)
= appear docked, extend out onto surface
Membranes surrounding bacterium start to look like rough ER (studded with ribosomes)
Within this ER-like compartment
= bacterium replicates
= eventually lyses host cell
OR
= default pathway of trafficking a non-pathogen
= bacterial uptake, then membrane-bound compartment acquires character of endosomes
= Dot / 1cm = defect in organele trafficking / intracellular multiplication
What does L. pneumophila do in the macrophage? What does it escape?
= can escape default endo-lysosomal pathway
(with help from effector proteins - infected into host cell through Dot/1cm type IV secretion system - T4SS)
= proteins + transport vesicles recruited to LCV (legionella containing vacuole)
= LCV slowly transformed to mimic ER = supports replication of L. pneumophila
= eventually host cell lysed = L. pneumophila released to infect other macrophages
What is the alarmone involved in the life cycle of L. pneumophila in protozoa and human macrophages?
ppGpp
= guanosine pentaphosphate or tetraphosphate
= an alarmone
= involved in stringent response in bacteria
= causes inhibition of RNA synthesis when there is a shortage of a.a present
What are the different ppGpp synthases?
RelA + SpoT
= allows L. pneumophila to sense its metabolic state
RelA
= detectes a.a. starvation
SpoT
= monitors disturbances in fatty acid synthesis
When nutrients become limited
= ppGpp accumulates in bacteria
= production of alternative sigma factor: RpoS
RpoS
= alternative sigma factor
= governs transition from replicative to transmissive form
= regulates the two component or quorum sensing systems (CpxRA, PmrAB, LetAB + LqsRS)
= which control metabolism / replication, motility, virulence traits
CrsA
= RNA-binding global regulator
= controls biphasic switch as an antagonist of the two component + quorum sensing systems
How do Amoeba act as training grounds for pathogens?
Many protozoan hosts allow intracellular bacterial replication
In outbreaks of Legionnaires’ disease
= amoebae and bacteria have been isolated from the source of infection
= isolated amoebae supported intracellular replication of the pathogen
Following intracellular replication with protozoa
= more resistant to chemical disinfection + biocides (compared to in vitro grown bacteria)
Protozoa shown to release vesicles of respirable size
= contain numerous L. pneumophila isolates
Vesicles are resistant to freeze-thawing + sonication
= bacteria within are highly resistant to biocides
Following release from protozoan host
= bacteria exhibit dramatic increase in infectivity for mammalian cells in vitro
= demonstrated intracellular bacteria that is more infectious
Number of bacteria isolated from source of infection of Legionnaire’s disease
= usually v. low or undetectable
= enhanced infectivity of intracellular bacteria within protozoa may compensate for low infectious dose
Viable BUT non-culturable L. pneumophila can be resuscitated by co-culture with protozoa
= observation may suggest failure to isolate is due to ‘dormant’ phase
No documented cases of bacterial transmission between individuals
= only source is environmental droplets from manmade devices (e.g. AC, shower heads)
