Lecture 31: Microbial growth & replication

Question 1

Bacteria can exist as planktonic cells, but usually live in

Answer 1

Biofilms

Question 2

(Planktonic-individual)

Answer 2

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.

Question 3

Stages of Biofilm Formation

Answer 3

Initial Attachment
Irreversible attachment:
Maturation 1
Maturation 2
Dispersal

Question 4

Initial Attachment

Answer 4

Individual bacteria attach weakly to a surface

Question 5

Irreversible attachment:

Answer 5

Attachment becomes irreversible using fimbrae and pilli.

Bacteria multiply and also attract other microbes to attach.

Question 6

Maturation 1

Answer 6

Bacteria secrete a sticky, protective Extra Cellular Matrix (ECM)- Polysaccharides, protein and DNA.

Question 7

Maturation 2

Answer 7

Biofilm grows in size and structure- form large 3D colony.

Question 8

Dispersal

Answer 8

sections of the biofilm break off.
The cells can go and colonise new areas.
Bacteria are 1000x more resistant to antibiotics in a biofilm

Question 9

Living in a biofilm is one of the most successful ways of living
Protects against:

Answer 9

Phagocytosis
Antibiotics
Disinfectants

Question 10

Biofilms are a huge problem in health care:

Answer 10

growth on medical devices and implants
Eg catheters, hip replacement, heart valve, stents etc

Question 11

biofilms in medical devices- urinary catheter

Answer 11

Usually a single species.
Example:
E. coli – common cause of Catheter associated urinary tract infection

Question 12

biofilms- heart valves

Answer 12

Endocarditis:
Infection of the heart valve.
Common cause Enterococcus sp
Eg Enterococcus faecalis

Question 13

biofilms- lung tissue

Answer 13

Cystic fibrosis patients.
Opportunistic infection by
Pseudomonas aeruginosa

Question 14

factors affecting growth of bacteria

Answer 14

nutrients,
iron,
oxygen
temperature
Ph

Question 15

Factors affecting growth: nutrients

Answer 15

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

Question 16

Factors affecting growth: Iron

Answer 16

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

Question 17

Bacterial iron transport system: are called

Answer 17

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

Question 18

Factors affecting growth: Oxygen- Aerobe

Answer 18

Aerobe
Requires oxygen for growth e.g. Mycobacterium tuberculosis

Microaerophile
Can grow in low concentrations of oxygen
e.g. Streptococcus pyogenes

Question 19

Factors affecting growth: Oxygen- Anaerobe

Answer 19

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

Question 20

Why is C. difficile a problem in hospital?

Answer 20

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:

1. 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).
2. 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.
3. 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.
4. 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.
5. Environmental Factors: Hospitals can be challenging environments to control infection due to the presence of numerous patients, high traffic areas, and shared facilities.
6. 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.

Question 21

Psychrophiles

Answer 21

Factors affecting growth: Temperature
-40oC to 20oC.
Optimum < 15oC
e.g. Listeria
monocytogenes

Question 22

Thermophiles

Answer 22

Factors affecting growth: Temperature
45oC to 100oC
e.g. Thermophilus
aquaticus

Question 23

Mesophiles

Answer 23

Factors affecting growth: Temperature
20oC to 40oC.
organisms of medical & pharmaceutical importance

Question 24

Factors affecting growth: pH

Answer 24

Most organisms of medical importance are
Neutrophiles: - grow best at pH 6.5-7.5

Question 25

Bacterial culture-Suspension

Answer 25

bacteria grown in complex liquid media as batch culture

To determine growth rate / effect of antimicrobial agents

Question 26

Bacterial culture- Colony

Answer 26

bacteria grown on complex media solidified with agar
To obtain pure culture / perform a viable count /assess diversity / aid identification

Question 27

Bacterial replication: Binary fission

Answer 27

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

Question 28

Microbial Growth Dynamics

Answer 28

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

Question 29

Persister cells

Answer 29

are known as Viable but non-culturable cells

Question 30

Microbial Growth Dynamics- exponential phase

Answer 30

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

Question 31

Microbial Growth Dynamics-death phase

Answer 31

Decline in cell numbers.
Some cells called persister cells don’t die

Question 32

Microbial Growth Dynamics-stationary phase

Answer 32

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

Question 33

Calculation of generation time (g)

Answer 33

Generation time is time required for cells to double in number
Can be read from exponential phase of growth curve

Question 34

Calculation of generation time (g)- During exponential growth:

Answer 34

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

Question 35

Direct measurement of bacterial number: Viable count

Answer 35

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

Question 36

Indirect measurement of bacterial number:
Optical density (OD)

Answer 36

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