L6 - Biofilms and antimicrobial tolerance

Question 1

Define the clinical significance of biofilms

Answer 1

Biofilms are matrix-enclosed consortia of microorganisms that are attached to biotic or abiotic surfaces

Although ubiquitous in nature, biofilms play a critical role in human health, ‘sheltering’ bacteria from antibiotics & host defence

Percentage of bacterial infections associated with biofilms varies from 65-80% depending on the report

P. aeruginosa is the most widely-studied organism in biofilm studies

Question 2

Describe the initial attachment & microcolony formation of biolfilms

Answer 2

• sad genes refer to ‘surface attachment defective’ mutants (i.e. genes that, when mutated, result in impaired surface attachment)
• sad genes generally fall into two distinct classes: motility-associated genes (including flagella) and Type IV pili genes

Type IV pili are associated with a type of surface-associated motility called ‘twitching motility’, which plays a role in the migration and aggregation of cells into microcolonies and the subsequent development of those into mature biofilms

• Significant overlap between factors required for attachment and those required for later (proliferative) stages of biofilm development
• In vitro, biofilms develop characteristic mushroom-shaped structures and open voids
• Open voids facilitate circulation of nutrients, oxygen etc.
  Mushroom-shaped structures are composed of a stalk and a cap
• Clonal expansion of bacteria make up the stalk (base) of the mushroom-like structure, whilst the mushroom ‘cap’ is mixed.
• The caps of the mushroom-like structures are formed by motile bacteria which ‘climb’ the stalks using type IV pili

Question 3

Klausen et al. (2003) studied the dynamics of biofilm development using fluorescently tagged strains of P. aeruginosa:

Answer 3

The left hand panel shows biofilm formed by a 1:1 mixture of YFP- and CFP-tagged wildtype. The stalk (base) of the mushroom is clonal (i.e. all the one colour), whilst the cap of the mushroom is mixed (blue & yellow). This indicates that the stalk is generated by clonal expansion, but that another mechanism is responsible for cap formation.

The panel on the right shows biofilm formed by a 1:1 mixture of YFP-tagged wildtype and a CFP-tagged pilA mutant that cannot make Type IV pili. It shows that type IV pili-mediated motility is required for cap formation, as the caps are exclusively yellow (i.e. comprised of the YFP-tagged wildtype).

Question 4

Describe the dispersal of biofilm communities

Answer 4

• Dispersal of cells or small aggregates from the biofilm can occur
• Gene regulatory networks may promote mobilisation of cells
• Shear forces can cause physical detachment of aggregates
• Dispersal can have profound clinical implications
  Facilitates spread throughout the organ or colonization of other sites.
  Detachment from catheters/implants may result in acute bloodstream infections or endocarditis

Question 5

Name the components of the biofilm matrix

Answer 5

Key components of the biofilm matrix are:
- Polysaccharides (in Pseudomonas: alginate, Pel & Psl)

  - Proteins
Extracellular DNA (eDNA)

• The precise composition of the matrix is influenced by environmental conditions, including nutrients and solid support

Question 6

Pseudomonas matrix components - alginate

Answer 6

• Alginate is responsible for the mucoid phenotype of P. aeruginosa
• It is not required for biofilm formation, but contributes to enhanced biofilm structure and architecture
• The alginate polysaccharide is responsible for the mucoid phenotype of P. aeruginosa. Production of alginate is not required for biofilm formation, but alginate over-producing strains will exhibit highly structured biofilm architecture.
• In contrast, production of the Pel and Psl polysaccharides does NOT results in a mucoid phenotype, but they play a more critical role in biofilm formation than alginate. Strains that are deficient in either Pel or Psl show impaired biofilm formation.

Question 7

Pseudomonas matrix components - Psl

Answer 7

• Psl polysaccharide
  Critically important for initial adherence to biotic and abiotic surfaces
  Also contributes to structural stability and architecture in mature biofilms

In contrast to alginate, the production of the Pel and Psl polysaccharides does NOT results in a mucoid phenotype, but they play a more critical role in biofilm formation than alginate. Strains that are deficient in either Pel or Psl show impaired biofilm formation.

Question 8

Pseudomonas matrix components - Pel

Answer 8

Pel polysaccharide
Like Psl polysaccharide, Pel is critical for biofilm formation; however, Pel is required for mature biofilm formation – not initial attachment.

• Data from Friedman & Kolter (2004):

Crystal violet staining is a commonly used method to assess biofilm formation. After allowing the biofilm to form, you wash off loosely-attached bacteria and then stain the remaining biomass with crystal violet. The image top left shows the result. To quantify the level of crystal violet staining, you then destain the biomass with a solvent (ethanol or DMSO). This liberates the crystal violet into solution, and you can measure the optical density.

The pel mutants are unable to form biofilms. This is not due to any defect in the initiation of biofilms, as shown in the graph bottom left. pelA, pelB, pelD & pelG all initiate biofilm formation essentially as the wildtype strain. The flgI mutant is included as a non-Pel control. FlgI is required for biofilm initiation.

The graph on the right then shows how those biofilms develop over time. This graph again shows that the initiation of biofilm is not impeded, but that the maturation of the resulting biofilm is.

Question 9

Pseudomonas matrix components - eDNA

Answer 9

• Extracellular DNA (eDNA) is a major matrix component
• In vitro, eDNA primarily arises through lysis of a subpopulation of cells
• In vivo, eDNA may also arise from lysis of host cells at the site of infection
• rhDNase is a therapeutic strategy used in CF

rhDNase – recombinant human deoxyribonuclease

The image above shows the ability of DNaseI to disperse young and mature biofilms. DNaseI was added to biofilms that were 12, 36, 60 or 84 hours old. DNaseI could cause the dispersal of young biofilms (12, 36 & 60 h) but not the mature biofilms at 84h.

• eDNA is not evenly distributed throughout the matrix: Type IV pili bind DNA with high affinity
  Proposed that the high concentration of eDNA in the outer region of the stalk cause accumulation of migrating bacteria, promoting cap formation
• Harmsen et al. (2010). FEMS Immunol Med Microbiol:
  The images of the biofilm above were generated by studying GFP-expressing P. aeruginosa and then staining he biofilm with the DNA-binding dye, propidium iodide (red). Propidium iodide is unable to enter cells as it is membrane impermeable. Consequently, it only stains eDNA. The left hand image shows a vertical section through a mushroom-like biofilm structure, with the right hand image showing a horizontal section through the stalk.

eDNA is concentrated in the outer region of the stalk, forming a border between the stalk subpopulation and the cap subpopulation.

Question 10

Pseudomonas matrix components - proteins

Answer 10

• Protein components facilitate surface adherence, and can promote matrix stability through interaction with other matrix components
• For example, CdrA binds carbohydrates, and specifically interacts with Psl
• Similarly, lectin proteins of P. aeruginosa (LecA & LecB) bind carbohydrates
• Other protein components relate to pili & flagella

The images show GFP-expressing Pseudomonas forming biofilms (both top-down and side-on views). The cdrA mutant on the right forms flatter and less structured biofilms compared to the wildtype (PAO1) on the left.

Question 11

Define QS and biofilm formation

Answer 11

Biofilm depth and density were assessed in wildtype P. aeruginosa and relevant QS-deficient mutants (deficient in AHL-based QS). This study specifically implicated the LasIR QS system in biofilm formation. LasI mutants had reduced biofilm depth and increased cell density (the smaller the distance to the cell’s “nearest neighbour”, the higher the density of the biofilm). Remember that biofilms need spaces and voids between cells in order to allow flow of nutrients etc. The deficiency in biofilm formation observed with the lasI mutant could be reversed by adding autoinducer.

• Both the AHL-based & PQS-based quorum sensing systems of P. aeruginosa contribute to eDNA release
  The PQS-deficient strain is also unable to form structured biofilms

Question 12

Describe the RhlIR QS system & rhamnolipid production

Answer 12

The RhlIR QS system of P. aeruginosa controls genes encoding rhamnolipid production (rhlAB)

Glycolipid biosurfactants (rhamnose head group and fatty acid tail)

Rhamnolipids contribute to biofilm architecture, specifically ensuring cell-free channels around macro-colonies

Rhamnolipids also promote release of planktonic cells at dispersal stage

Surfactants are compounds that lower the surface tension between two liquids or between a liquid and a solid.

Question 13

c-di-GMP: A critical regulator of biofilm formation

Answer 13

Cyclic-di-GMP, c-di-GMP, is a nucleotide-based molecule that acts as a major secondary messenger within the bacterial cell. It favours biofilm formation, whilst suppressing motility & acute virulence.

Balancing c-di-GMP levels:
- C-di-GMP levels are controlled by the actions of two types of enzymes:

• Diguanylate cyclases (DGCs)
  Contain a GGDEF domain
• Phosphodiesterases (PDEs)
  Contain either EAL or HD-GYP domains
• The genome of P. aeruginosa encodes approximately 40 proteins that contain either a DGC, a PDE or both GGDEF & EAL domains
• In most cases, detail of the mechanisms and stimuli controlling DGC/PDE activity remain to be defined

Question 14

Describe the antimicrobial tolerance in biofilms

Answer 14

Unlike antibiotic resistance, tolerance is a transient non-heritable phenotype

Bacteria in biofilms can be up to 1,000 times more tolerant to antibacterials than equivalent planktonic bacteria, and biofilm formation is an important reason for failure of antimicrobial therapy

Tolerance mechanisms associated with biofilms include:
Reduced metabolic activity and growth rates
Presence of dormant persister cells
Restricted diffusion of antimicrobials through the biofilm matrix

Question 15

Restricted diffusion is antibiotic & biofilm specific

Answer 15

Most antimicrobial agents can readily diffuse into the biofilm matrix
Slowed diffusion may enable microbes to mount an adaptive response

Diffusion is profoundly influenced by charge and specific interactions with matrix components
- Positively-charged aminoglycoside antibiotics bind alginate; negatively-charged β-lactam antibiotics do not.

• Similarly, in non-mucoid P. aeruginosa biofilms, tobramycin is sequestered in biofilm periphery via Psl &/or Pel polysaccharides

Question 16

Define the physiological heterogeneity within biofilms

Answer 16

As most antibiotics target metabolic processes, presence of bacteria with low metabolic activity in biofilms is a critical determinant of tolerance.

Considerable heterogeneity exists within biofilm populations. Concentration gradients arise through differing metabolic activities of bacterial cells coupled with variable diffusion properties through the biofilm matrix that influences concentrations of nutrients, signalling molecules, bacterial waste products, oxygen etc. Cells deep within biofilms typically have very low metabolic activity, which has significant implications for eradication of biofilm-associated infections.

Question 17

The influence of metabolic activity on tolerance

Answer 17

Tetracycline and ciprofloxacin target metabolically-active bacteria (targeting DNA replication and translation respectively). They only kill cells within the biofilm periphery. In contrast, colistin interferes with the membrane structure irrespective of metabolic activity. Metabolically-active cells in the periphery were able to adapt to colistin challenge (presumably by modifying their LPS) and so were not killed. In contrast, the metabolically-inactive cells deeper within the biofilm were killed by colistin.

Question 18

Describe ‘Persister cells’ and antimicrobial tolerance

Answer 18

First identified in the 1940s, ‘persister cells’ are dormant variants of regular cells that are highly tolerant to antibiotics

The frequency of persister cells is highest in stationary phase cultures

Question 19

Persister cells & biofilm drug tolerance

Answer 19

Persister cells have been detected in biofilms, leading to a model of relapsing biofilm infection.
Persister cells are antibiotic tolerant – not antibiotic resistant. In other words, they are not killed by the presence of antibiotics, but they do not grow in the presence of antibiotics either. They will only resume growth once the antibiotics is removed. However, at that point, they become fully susceptible to the antibiotic again (i.e. it is not inherited resistance).

Question 20

Describe the Toxin-antitoxin (TA) loci & persister cells

Answer 20

Bacterial genomes contain toxin – antitoxin loci (TA loci)
Toxin inhibits cellular functions within the bacterial cell
Anti-toxin ordinarily represses the toxin.

• The anti-toxin is more susceptible to proteolysis than the toxin, thus altered proteolytic activity can shift the balance towards free toxin
• Factors that govern the activity of TA loci and/or persister cell formation remain poorly defined

Question 21

Proving the link between TA loci & persisters

Answer 21

Typically, linkage of genes to specific phenotypes is achieved by mutagenesis
However, knockout libraries have not identified mutants lacking persisters, indicating redundancy in dormancy mechanisms
The role of TA loci has typically been investigated by overexpressing the toxin and assessing impact on growth & persister frequency.

• Data from Butt et al. (2014). Biochem J. 459, 333-344:

In the above set of experiments, a toxin from a putative toxin-antitoxin locus of Burkholderia pseudomallei was expressed in E. coli under the control of an arabinose-inducible promoter. Inclusion of glucose in the media inhibits the expression of the toxin, whilst inclusion of arabinose induces expression of the toxin (the level of expression being proportional to the concentration of arabinose). The graphs show the frequency of persister cells obtained with and without toxin induction following treatment with either ciprofloxacin (active against DNA replication) or ceftazidime (a beta-lactam antibiotic active against the cell wall). Toxin induction significantly enhanced persister frequency. It is also notable that there are different persister frequencies achieved with different antibiotics, suggesting different populations of persister cells.

Question 22

Increasing persisters during chronic infection?

Answer 22

Reported emergence of hip (high persister) mutants in late-stage CF isolates.

• Data from Mulcahy et al. (2010). J. Bacteriol. 192, 6191-6199.:
  The graph shows the persister cell frequency of 14 pairs of isolates from 14 individual patients (each AMT number is a different patient). Within each pair of isolates, we are looking at an early isolate and a late isolate (white bars and black bars respectively). In 10 of the 14 patients, the late isolate forms a significantly higher frequency of persister cells than the early isolate. Such mutants are generally referred to as hip mutants (high persister mutants), although the molecular basis for the high level of persistence is not characterized.

Question 23

Conclusions

Answer 23

Biofilms are ubiquitous, and play a critical role in infections

Biofilms are highly-structured microbial communities encased within a matrix consisting primarily of polysaccharides, eDNA & proteins

The biofilm matrix provides protection against the immune system and against antibiotics

Multiple mechanisms contribute to the antibiotic tolerance of bacteria within biofilm communities