This course will give you an introduction to bacteria and chronic infections. Leading experts in the field will make you familiar with the fundamental concepts of microbiology and bacteriology such as single cell bacteria, biofilm formation, and acute and chronic infections.
2.4 Chronic Infections - Treatment failure by Associate professor Oana Ciofu
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En provenance du cours de University of Copenhagen
Bacteria and Chronic Infections
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Properties of Biofilms and Chronic Infections
Hello everyone, welcome to the second module. During this module you will learn about the specific properties of biofilms and chronic infections.
Cheers Thomas
Rencontrer les enseignants
Thomas BjarnsholtProfessor
Costerton Biofim Centre
Hi, my name is Oana Ciofu and I am an associate professor of Medical Microbiology at the Faculty
of Health and Medical Sciences at University of Copenhagen.
In the lecture, today, I will focus on the mechanisms involved in the tolerance of biofilms
to antibiotics. Which implications the biofilms tolerance to antibiotics has for the treatment
of chronic infection will be presented in a separate lecture.
The tolerance of biofilms to antibiotics is part of the definition of biofilms.
Biofilm of all bacterial species, are tolerant to all types of antibiotics
The tolerance develops together with the biofilm: freshly formed, young biofilms are much more
susceptible to antibiotics than 3 to 5 days later as shown by the increased concentration
of antibiotic needed to eradicate old biofilms compared to young biofilms.
The tolerance of bacteria in biofilms to antimicrobial compounds is multifactorial, being a result
of an interplay between three types of tolerance mechanisms: the physical, the physiological,
and the adaptive tolerance mechanisms. Physical tolerance depends on the three-dimensional
structure of the biomass consisting of bacterial cells and matrix. Some of the matrix components
such as DNA and polysaccharides (alginate) binds some types of antibiotics such as tobramycin
and colistin . This bindings leads to a delayed diffusion of the antibiotic molecules in biofilm,.
Therefore, biofilms formed by mucoid, alginate hyperproducer P. aeruginosa are more difficult
to eradicate than biofilms formed by non-mucoid strains. Here you can see, that mucoid biofilms
(PDO300) are more difficult to treat than non-mucoid (PAO1).
Thus, factors regulating the matrix formation play a role in the physical tolerance of biofilms.
Antibiotics at sub-inhibitory concentrations can increase formation of the biofilm, mainly
through increased matrix formation and in this way might increase the tolerance to the
antibiotic molecules. Another factor influencing the tolerance is
the quorum-sensing which is a cell-cell communication mechanism that synchronizes gene expression
in response to population cell density The involvement of quorum sensing can be explained
through its role in the production of extracellular DNA which inhibits penetration of some antibiotics
into the biofilm. The extracellular DNA that is produced in P. aeruginosa biofilms also
has a stabilizing effect. Because biofilms formed by P. aeruginosa quorum-sensing mutants
are more fragile than wild type biofilms they may be more vulnerable to for example shear
forces and phagocytosis, and because of the high bacterial turnover a large proportion
of the bacteria will be growing actively and may therefore show increased sensitivity to
some antibiotics. Here, you can see that tobramycin is more
efficient in killing a P.aeruginosa mutant that is not producing quorum-sensing (ΔlasRrhlR)
compared to a QS producing strain (PAO1). Physiological tolerance is determined by the
metabolic state of the bacteria growing in biofilms.
Here the metabolic rate is represented by the protein synthesis ability which is present
only at the air-liquid interfase of the biofilm. The growth rates of the inner layers of the
biofilm decreases with increasing limitation of nutrients and oxygen. Here you can see
that the oxygen concentration is decreasing fast from the top of the biofilm, reaching
anaerobic conditions at a depth of only 50µm. It has been shown in vitro, that biofilms
consist of at least two distinct subpopulations: a growing, aerobic subpopulation, and a more
dormant and anaerobic growing subpopulation. Antibiotics acting on bacterial membranes-such
as colistin—are effective at low oxygen concentrations and therefore will be able
to kill the inner part of the biofilm. Antibiotics such as aminoglycosides, fluoroquinolones
and beta-lactams do not function well under low oxygen tension and therefore kill only
the outer, growing part of the biofilm (panelA). A combination of these two drugs will have
a synergistic effect (panel B). A similar effect is seen when colistin is
combined with tobramycin . The killing effect of a combination therapy
of aminoglycosides, fluoroquinolones and colistin is, however, not complete as a small fraction
of bacteria always survive, probably owing to the presence of persisters.
Persisters are phenotypic variants of regular cells described as non-growing, dormant, multi-drug
tolerant representing a small bacterial subpopulation (< 0.1%) that survives antibiotic treatment
of biofilms. A large number of different genes have been shown to be involved in the occurrence
of the persister phenotype, suggesting that multiple pathways can lead to the persister
cell phenotype. “Waking-up” the persisters by stimulating
their metabolism, has been proposed as a potential therapeutic approach to treat biofilms.
Adaptive tolerance or transient tolerance of biofilm refers to the transient induction
of resistance mechanisms only in the presence of antimicrobials..
When the antibiotic is eliminated and the antibiotic exposure stops, this type of tolerance
disappears. It does not require genetic changes, and is
mediated by regulatory networks that are switched on only in the presence of antibiotics.
The adaptive tolerance can be either antibiotic-specific or non-specific.
Examples of specific tolerance mechanisms are beta-lactamase induction (enzymes that
inactivate beta-lactams) in the presence of beta-lactams or alteration of lipopolysaccharide
on the outer membrane of Gram-negative cells (LPS) in the presence of colistin.
Examples of non-specific tolerance mechanisms are: up-regulation of efflux pumps, conversion
of wild-type sensitive bacterial cells to tolerant rough small colony variants or protection
against drug-induced oxidative stress. At first sight, these different mechanisms
involved in the tolerance of biofilms to antibiotics might seem that they act independently, however
there is evidence in P. aeruginosa biofilms of common regulators (like the transcriptional
regulator brlR) that is involved in the tolerance against oxidative stress and regulation of
efflux-pumps expression. I will discuss in more detail some of these
mechanisms involved in adaptive tolerance.
Protection against drug-induced oxidative
stress, a non-specific tolerance mechanism It has been shown that the killing mechanism
of bactericidal antibiotics involves perturbation of the bacterial metabolism leading to formation
of reactive oxygen species (ROS) that can either be lethal or mutagenic for the bacteria,
depending on the concentration. This killing mechanism was shown to be involved
in the treatment of P. aeruginosa biofilms with ciprofloxacin, as well as in the killing
of Burkholderia cenocepacia biofilms with tobramycin and of Candida albicans with miconazol
indicating that ROS production is a general mechanism involved in the killing of bacteria
and fungi. Protection against oxidative stress decreases the activity of bactericidal and
fungicidal drugs and the mechanisms that provide this protection are important for survival
of treated biofilms.. In P. aeruginosa biofilms the protection against
ROS is controlled by a regulatory system involved in response to nutrient limitation called
stringent response (SR). The SR protects P. aeruginosa against antibiotic-induced oxidative
stress by maintaining adequate levels of protective enzymes against ROS ( catalase, superoxide).
So, stringent response seems to play an important role in protection of biofilms against antibiotic
induced-oxidative stress. Inactivation of SR can, thus, be a possible mechanism for
antibiofilm drugs.
Changing the composition of the outer membrane
(LPS), a specific tolerance mechanism against colistin
Colistin has to reach the cytoplasmic membrane of the Gram-negative bacteria and therefore
it has to pass through the cell wall binding to the lipopolysaccharide (LPS) molecule on
the outer membrane.
In response to the presence of the antibiotic, the bacterial cells are through regulatory
systems (such as PmrA-PmrB two-component regulatory systems) changing the LPS molecule by adding
an arabinose. As this mechanism requires energy, it is present only in the metabolically active
subpopulations that survive, as you can see in the picture the antibiotic treatment.
3. Induction of beta-lactamase in biofilms, a specific tolerance mechanism against beta-lactam
antibiotics In P. aeruginosa and other Gram-negatives,
the production of beta-lactamase, an enzyme that inactivates the beta-lactam antibiotics
is inducible in the presence of the antibiotic and disappear when the concentration of the
antibiotic decreases. The various beta-lactams have various beta-lactamase induction capacities.
Bacteria can acquire mutation in the complex regulatory system of beta-lactamase induction
and they can become stable producers of beta-lactamase and therefore resistant to this important
group of antibiotics. In this experiment the promoter region of
the structural gene of beta-lactamase was tagged with green fluorescence protein and
biofilms were formed in flow-cells. In this picture you can see that under treatment with
ceftazidime which is a weak inducer of beta-lactamase, the enzyme is induced in the superficial layer
of the biofilm (in green). However, when the P. aeruginosa biofilm is
treated with imipenem , which is a strong inducer of beta-lactamase, the enzyme is induced
in all the biofilm layers ( in green). The whole biomass of the biofilm in red.
The presence in the biofilm matrix of beta-lactamases will lead to the hydrolysis of the beta-lactam
antibiotics before they reach the bacterial cells at the bottom of the biofilm. Nichols
(Nichols et al. 1989) predicted from mathematical models that bacteria expressing high levels
of chromosomal beta-lactamase growing in biofilms would be exposed to reduced concentrations
of beta-lactam antibiotics due to accumulation of the enzyme in the polysaccharide matrix.
Beta-lactamase can be excreted in membrane vesicles and entrapped in the biofilm matrix
impairing the penetration of the beta-lactam molecules into the biofilm (Ciofu et al. 2000).
This has implication for the dosage and mode of administration of the beta-lactam antibiotics
as it will be presented later. In contrast to tolerance, antibiotic resistance
is heritable, due to mutation and is not reversible. Development of antibiotic resistance in biofilms
even in the absence of antibiotic selection has been reported confirming that biofilms
are a fertile ground for development of resistance. Evolution of resistance involves mutations
that convey resistance to single or to several antibiotics.
The persistence in biofilm of these mutations depends on their fitness cost for the survival
in the compartmentalized structure of biofilms. The occurrence of variants resistant to antibiotics
provides an “insurance effect” by creating subpopulations of cells that can survive or
even proliferate when the biofilm come under antibiotic assault facilitating reconstitution
of bacterial biofilm populations after cessation of the antibiotic treatment.
A mechanism for the increased mutability in biofilms is oxidative stress. This is due
to an increased production of endogenous reactive oxygen species (ROS) and a deficient anti-oxidant
system in biofilms. It is important to stress that this biofilm-intrinsic oxidative stress
is antibiotic independent and not related to the ROS formation previously described
under antibiotic treatment of biofilms. The notion of oxidative stress in biofilms
might be paradoxical given the low oxygen tension detected deep within biofilm structures,
however, respiration can produce enough oxidative stress to produce DNA damage.
In addition to the endogenous oxidative stress, the biofilm-growing bacteria in CF airways
are exposed to exogenous ROS from the activated immune cells which surround the biofilm.
There is hypoxic environment in the CF sputum due to the consumption of oxygen by PMNs which
liberate reactive oxygen species that can react with the biofilm-embedded bacterial
cells thus creating a unique environment in the sputum with low-oxygen tension filled
with reactive oxygen species. An association between oxidative stress and
the occurrence of hypermutable P. aeruginosa strains was shown in CF patients
The increased mutability in biofilms promotes the emergence of mutations including mutations
conferring antibiotic resistance. The antibiotic resistant mutants will be selected for by
the repeated antibiotic courses administered as shown in the following competition experiment.
After only 4 days of ciprofloxacin treatment (t2) the mutator , blue strain took over the
whole biofilm structure, while in the control (untreated, 4-day-old) mixed biofilms most
of the structure was formed by the PAO1 strain. Interestingly, the proportion of blue hypermutators
seemed to be increased at t4 (6-day-old mixed biofilm) in control biofilms, suggesting that
biofilm growth itself favours the selection of mutator populations.
Development of mutational resistance to all classes of antibiotics during the chronic
biofilm lung infection in CF has been documented. It has also been shown that hypermutable strains
form better biofilms than strains with normal mutation frequency. Mutants in DNA repair
such as PAO1 ∆mutT strains formed biofilms with significantly enhanced microcolony growth
compared to both the wild-type and the respective complemented strains. Biofilms created by
the hypermutator strains were significantly larger in total biovolume and maximum microcolony
thickness.
When this P. aeruginosa biofilm with increased biomass was treated with piperacillin/tazobactam,
it survived better to the treatment compared to the wild-type biofilm. The better survival
can be explained by increased tolerance due to physical mechanisms, such as increased
biomass or due to selection of resistance mutants, or a combination of both mechanisms.
As you can see the tolerance of biofilms to antibiotics is multifactorial and it insures
persistence of the bacterial biofilms which can become resistance due to mutations increasing
the recalcitrance of biofilms to antibiotics. This represents an important challenge for
the treatment of biofilm infections, as it will be discussed later.
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