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.
