The document discusses biofilms and their antibiotic resistance. It notes that biofilms are assemblages of surface-attached microbial cells encased in an extracellular matrix. This matrix provides protection from antibiotics by acting as a diffusion barrier and binding antimicrobial agents. Additionally, the heterogeneous conditions within biofilms, including nutrient depletion and slower growth, contribute to antibiotic tolerance in some cells. Genetic transfer of resistance genes is also facilitated within dense biofilm communities.

1. OmidTeymournejad
(Medical Bacteriology, PhD)

2.  A biofilm is an assemblage of surface-
associated microbial cells that is enclosed in
an extracellular polymeric substance matrix
 Biofilms may form on a wide variety of
surfaces, including living tissues, indwelling
medical devices, industrial or potable water
system piping

3.  Biofilms are a major cause of human
infections
 The majority of hospital-acquired infections
are due to biofilms because they can be life-
threatening colonizers of biomedical devices

6.  The first step is the attachment of the
bacterial cells to the selected abiotic or biotic
surface
 Initial attachment is mediated through weak
reversible van derWaals interactions
between the cell surface and the substratum,
which can lead to stronger adhesion receptor
mediated attachment

7.  Bacterial cell surface structures such as flagella,
fimbriae, LPS, and exopolysaccharides
participate in irreversible interactions
 The second step corresponds to the
development of micro-colonies promoted by
the growth and division of the first attached
cells (primary colonizers)

8.  The micro-colonies progressively enlarge and
coalesce to form the first layer of cells
covering the surface
 When multiple layers of cells pile up on the
surface, the third step of the formation is
obtained

9.  indicated by the presence of a mature biofilm
characterized by the presence of macro-
colonies surrounded by water channels that
help distribute nutrients and signaling
molecules
 Finally, to survive when nutrients become
limited, or simply to spread and colonize to
other niches, some biofilm cells can detach
individually or in clumps

13.  biofilms display a common attribute, the biofilm
matrix.
 Contrary to free-floating planktonic cells, biofilm
cells are embedded in a self produced
extracellular matrix, the extracellular polymeric
substance (EPS), that holds them together
 Biofilms are composed of about 80–85% EPS (by
volume) and only 15–20% cells (by volume)

14.  Although the EPS may vary in chemical and
physical properties, its major components are
polysaccharides, proteins, and extracellular
DNA
 The EPS plays a major role in maintaining the
integrity of the biofilm and can confer other
beneficial properties.

15.  Since the EPS is also highly hydrated, it can
prevent desiccation in some natural biofilms
 The EPS can also act as a diffusion barrier,
preventing toxic substances such as
antibiotics and disinfectants from reaching
their target

16.  Most biofilms found in nature are
polymicrobials, where diverse species
expressing multiple phenotypes are involved
 The most amazing fact is that even in a
mono-species biofilm, phenotypic
heterogeneity exists

17.  Cell differentiation in biofilms may depend on
the local environmental conditions
surrounding the cells
 Different concentration gradients of oxygen,
nutrients, ions, and chemicals create a wide
variety of microhabitats providing conditions
suitable for bacterial colonization

18.  Cells located in the upper biofilm layers
consume all available oxygen and grow
aerobically, while an anaerobic micro-niche
developed underneath the aerobic layer
 Oxygen- and nutrient depleted regions are
found at the bottom layers of the biofilm
structure and under these circumstances,
most of the sessile cells are metabolically
inactive or dead

19.  A few years ago,Watnick & Kolter proposed
the interesting idea that biofilms can be
compared to cities
 In these cities of microbes, microorganisms
are considered “social” organisms able to
communicate with one another

20.  Using different chemical languages, bacteria
learn about their current cell population and
determine when they have reached a critical
mass
 Using that information, bacteria can thus
modify their behavior to carry out processes
that would require many cells acting in
conjunction to be effective

21.  Cell-to-cell communication is generally
carried out by diffusible signal molecules
produced and released by bacteria
 When bacteria are growing within a biofilm,
they secrete signaling molecules (auto-
inducers) that increase in concentration as a
function of bacterial cell density

22.  In a process called quorum sensing, bacteria
communicate with one another by using
auto-inducers to regulate their gene
expression in response to fluctuations in the
cell population density
 Two types of quorum-sensing systems are
recognized in bacteria:

23.  intra-species communication and inter-
species communication
 During intra-species communication, several
auto-inducers have been identified

24.  Gram-negative bacteria usually use acyl
homoserine lactone (AHL) as signal
molecules, while Gram-positive bacteria
utilize small peptides
 During inter-species communication, bacteria
use autoinducer-2 (AI-2)

25.  In some cases, quorum sensing does not
seem to be involved in biofilm structural
development
 while for other species, there is evidence that
quorum sensing is important for the
attachment of bacteria to the surface, the
maturation of the biofilm, or the control of
events leading to the dispersion of cells

26.  A significant characteristic of microbial
biofilms is their high-level drug tolerance
 Bacterial biofilms have been shown to have a
100- to 1,000- fold increased tolerance
toward antibiotics in comparison to their
free-swimming counterparts

27.  Diffusion barrier imparted by the EPS matrix
 Phenotypic heterogeneity
 GeneTransferring
 Secretion of antibiotic degrading enzymes

28.  A primary function that has been attributed
to the biofilm matrix is protection,
 Several studies have shown that the EPS
matrix can act as an impermeable barrier to
limit antimicrobial penetration, thereby
protecting the biofilm cells

29.  Such protection can be due either to physical
hindrance in antimicrobial diffusion or to
direct binding of the antibiotics by the EPS
matrix
 Upon antibiotic treatment, cells at the top of
the liquid– biofilm interface die due to their
closer exposure, while bacteria embedded
deep inside the biofilm are able to survive

30.  Anionic EPS matrix can bind and sequester
toxic cationic heavy metals, cationic
antimicrobial peptides, and positively-
charged antibiotics (e.g. aminoglycosides)

31.  Phenotypic heterogeneity occurs within
biofilms notably due to different
concentration gradients
 Cells localized at the bottom layers of the
biofilm are normally found in a growth stage
analogous to stationary-phase planktonic
cells, while the physiology of cells localized at
the top surface layers is similar to
exponentially growing planktonic cells

32.  Microbial cells, especially those in the deeper
layers of the biofilms where nutrients and
oxygen are limited, are associated with a
lower growth rate
 This reduced metabolic activity might
account for the enhanced tolerance toward
antibiotics that target bacterial cellular
processes such as DNA replication or
translation

33.  Conventional antibiotics used to treat
infections are mostly effective at killing
rapidly growing cells
 The decreased metabolic activity of cells
found within the deeper biofilm layers may
thus contribute to antibiotic tolerance and
the persistence of biofilm infections

34.  Biofilms also provide an ideal niche for the
exchange of extrachromosomal DNA
(plasmids)
 Conjugation (the mechanism of plasmid
transfer) occurs at a greater rate between
cells in biofilms than between planktonic cells