Lecture 31: Microbial growth & replication Flashcards
Bacteria can exist as planktonic cells, but usually live in
Biofilms
(Planktonic-individual)
Bacteria form Biofilms on any surface where there is moisture.
Bacteria attach to surface, grow, and become enveloped in an extra-cellular matrix (ECM) composed mostly of polysaccharides, proteins and DNA.
Stages of Biofilm Formation
Initial Attachment
Irreversible attachment:
Maturation 1
Maturation 2
Dispersal
Initial Attachment
Individual bacteria attach weakly to a surface
Irreversible attachment:
Attachment becomes irreversible using fimbrae and pilli.
Bacteria multiply and also attract other microbes to attach.
Maturation 1
Bacteria secrete a sticky, protective Extra Cellular Matrix (ECM)- Polysaccharides, protein and DNA.
Maturation 2
Biofilm grows in size and structure- form large 3D colony.
Dispersal
sections of the biofilm break off.
The cells can go and colonise new areas.
Bacteria are 1000x more resistant to antibiotics in a biofilm
Living in a biofilm is one of the most successful ways of living
Protects against:
Phagocytosis
Antibiotics
Disinfectants
Biofilms are a huge problem in health care:
growth on medical devices and implants
Eg catheters, hip replacement, heart valve, stents etc
biofilms in medical devices- urinary catheter
Usually a single species.
Example:
E. coli – common cause of Catheter associated urinary tract infection
biofilms- heart valves
Endocarditis:
Infection of the heart valve.
Common cause Enterococcus sp
Eg Enterococcus faecalis
biofilms- lung tissue
Cystic fibrosis patients.
Opportunistic infection by
Pseudomonas aeruginosa
factors affecting growth of bacteria
nutrients,
iron,
oxygen
temperature
Ph
Factors affecting growth: nutrients
Nutrients are required for cellular biosynthesis & energy generation
Macroelements:
C, H, O, N, water
S, P, K, Ca, Mg, Fe
Trace elements:
Mn, Zn, Co, Mo, Ni, Cu
vitamins & growth factors
Bacteria colonising /infecting our bodies derive nutrients from their host
Factors affecting growth: Iron
Crucial for growth of almost all bacteria; used for energy generation
Iron in body not ‘available’
- within mammalian cells:
90% stored in ferritin or as haem group
8% stored in other cellular proteins e.g. myoglobin
- outside mammalian cells: 1-2% attached to transporters:
e.g. transferrin in serum, lactoferrin in mucosal secretions
- Non complexed iron exists as Fe 3+ : insoluble; not absorbed into blood
Bacterial iron transport system: are called
Siderophores
Low molecular weight compounds with high affinity for iron
High energy strategy
Produced & exported from some bacteria when conc. of iron is low
Bind iron & allow uptake into the cell
- Remove iron complexed with transferrin. Enable uptake into bacterial cell
Factors affecting growth: Oxygen- Aerobe
Aerobe
Requires oxygen for growth e.g. Mycobacterium tuberculosis
Microaerophile
Can grow in low concentrations of oxygen
e.g. Streptococcus pyogenes
Factors affecting growth: Oxygen- Anaerobe
Does NOT require oxygen for growth
Obligate anaerobe
Cannot grow in the presence of oxygen
e.g. Clostridioides difficile
Facultative anaerobe
Can grow in oxygen if it is available
e.g. E. coli
Why is C. difficile a problem in hospital?
Clostridium difficile, often referred to as C. difficile, is a bacterium that can cause symptoms ranging from diarrhea to life-threatening inflammation of the colon. It is a problem in hospitals for several reasons:
- Antibiotic Use: Antibiotics disrupt the natural balance of bacteria in the gut, allowing C. difficile to multiply and cause infection. In hospitals, patients often receive antibiotics, which increases their risk of developing C. difficile infection (CDI).
- Contagiousness: C. difficile spores are highly contagious and can be spread through contact with contaminated surfaces or objects, as well as through the fecal-oral route. In a hospital setting where patients and healthcare workers are in close proximity, the risk of transmission is higher.
- Vulnerable Population: Hospitalized patients are often already in a weakened state due to illness or surgery, making them more susceptible to infections like C. difficile.
- Frequent Healthcare Contact: Hospitalized patients typically have multiple interactions with healthcare workers, who may inadvertently transmit C. difficile spores from one patient to another if proper infection control practices are not followed rigorously.
- Environmental Factors: Hospitals can be challenging environments to control infection due to the presence of numerous patients, high traffic areas, and shared facilities.
- Severity of Infection: In some cases, C. difficile infection can lead to severe complications such as pseudomembranous colitis, toxic megacolon, sepsis, or even death, particularly in vulnerable populations like the elderly or those with underlying health conditions.
Due to these factors, C. difficile represents a significant healthcare-associated infection problem in hospitals, leading to increased morbidity, mortality, and healthcare costs. Preventative measures, including strict antibiotic stewardship, adherence to infection control protocols, and environmental cleaning, are crucial in reducing the incidence and transmission of C. difficile in healthcare settings.
Psychrophiles
Factors affecting growth: Temperature
-40oC to 20oC.
Optimum < 15oC
e.g. Listeria
monocytogenes
Thermophiles
Factors affecting growth: Temperature
45oC to 100oC
e.g. Thermophilus
aquaticus
Mesophiles
Factors affecting growth: Temperature
20oC to 40oC.
organisms of medical & pharmaceutical importance
Factors affecting growth: pH
Most organisms of medical importance are
Neutrophiles: - grow best at pH 6.5-7.5
Bacterial culture-Suspension
bacteria grown in complex liquid media as batch culture
To determine growth rate / effect of antimicrobial agents
Bacterial culture- Colony
bacteria grown on complex media solidified with agar
To obtain pure culture / perform a viable count /assess diversity / aid identification
Bacterial replication: Binary fission
ONE bacterial cell grows & divides into TWO identical daughter cells
Cell elongates to approx. double length.
DNa replication -Cell about to divide copies its chromosome
Septum formation-A septum begins to form
The two copies of the chromosome are pulled apart.
Cell separation- Septum formation continues until two daughter cells are pinched off
Cell division continues until nutrients depleted or conditions become unfavourable
Cells can potentially divide forever
Microbial Growth Dynamics
Bacteria multiply by doubling as fast as conditions will allow;
- Exponential growth.
Time taken to divide is the Generation time
Rate of cell division determined by
(i) time required for DNA replication
(ii) conditions
Persister cells
are known as Viable but non-culturable cells
Microbial Growth Dynamics- exponential phase
The cells are behaving in a constant predictable way .
The generation time is constant
(straight line on our graph )
The ideal phase to use the bacteria for research
Microbial Growth Dynamics-death phase
Decline in cell numbers.
Some cells called persister cells don’t die
Microbial Growth Dynamics-stationary phase
The population is running out of resources
No increase or decrease in cell numbers-
- some cells are dividing , some are dying
The cells are behaving unpredictably
Calculation of generation time (g)
Generation time is time required for cells to double in number
Can be read from exponential phase of growth curve
Calculation of generation time (g)- During exponential growth:
NT = N0 x 2^n
Log10Nt= Log10N0 + nLog102
No = number of cells initially present
NT = number of cells present at time T
n = number of generations
Generation time (g) = Time (T) / no. of generations (n)
generation time in minutes
Direct measurement of bacterial number: Viable count
1 bacterium produces 1 colony (CFU) on agar plate which
can be counted
Dilute sample of bacteria
Spread on agar plate
Incubate overnight @ 37oC
Count colonies
VC expressed as Colony Forming Units / ml (CFU/mL)
Use standard form eg 5000 is 5x103
Best way to measure no. actively dividing cells
EXCEPT for clumps/chains of cells
Conditions must be suitable for growth
Requires overnight culture
Indirect measurement of bacterial number:
Optical density (OD)
Optical density increases with increasing cell no. over time
Cell no. directly related to OD.
Read off cell no. from standard curve of OD vs. cell number
Cells must be in exponential phase for OD to represent no. dividing cells
