Mechanistic insights in the antibiotic tolerance of Pseudomonas aeruginosa biofilms
Valentin, Jules
DOI:
10.33612/diss.160159324
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2021
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Valentin, J. (2021). Mechanistic insights in the antibiotic tolerance of Pseudomonas aeruginosa biofilms. University of Groningen. https://doi.org/10.33612/diss.160159324
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
1 Antimicrobial resistance
Chapter 1
Antimicrobial resistance
Emerged on Earth more than 3 billion years ago, microorganisms were the first life forms and have evolved to become indispensable actors in our environment. 1 Despite their importance
for the proper functioning of the human body and many processes in the natural world, a small percentage of microbes are pathogenic to humans and have been responsible for deadly pandemics, such as the Black Plague during the 14th century, the Spanish flu in the 20th century
and more recently the coronavirus disease 2019. 2,3 A key step towards better treatment was
the understanding of the link between microorganisms and infectious diseases. In 1676, Antony van Leeuwenhoek was the first to observe bacteria and called them dierkens (Dutch for little animals and translated to English as animalcules) (Figure 1). 4 Centuries later, in the
1860s, Louis Pasteur advanced the germ theory to explain infectious diseases and developed the first vaccines for fowl cholera, anthrax and rabies. 5 The discovery of penicillin by Alexander
Fleming in 1928 initiated the Golden Age of antibiotics through isolation of antibiotic-producing microorganisms, still in use in our modern society. 6 However, Alexander Fleming, during his
Nobel Lecture in 1945, warned that misuse of antibiotics would result in the selection of resistant pathogens. 7 As predicted, we are currently living in an antibiotic resistance crisis, in
which antimicrobial resistant (AMR) bacteria account for 700,000 deaths each year worldwide and are estimated to be responsible for 10 million deaths by 2050 if resistance is left unchecked. 8 Infectious diseases remain nowadays amongst the deadliest diseases in
developing countries, of which lower respiratory infections have been ranked the fourth most common cause of deaths worldwide in 2017. 9 Two main factors contribute to the AMR crisis:
overuse and misuse of antibiotics driving the emergence of resistant strains, and lack of development of new classes of antibiotics. 10
3 Antimicrobial resistance
Figure 1. Van Leeuwenhoek's microscope and drawings of bacteria extracted from the human mouth in 1683. 4 Van Leeuwenhoek's microscopes consisted of a highly polished double-convex lens mounted between two metallic plates (about 2 by 4 cm), and were able to magnify several hundred times with a resolution power of about 1 µm.
Biofilm formation
The emergence of new technologies brought light to the unexplored complexity of the microbial world. In the 1970s, improvements in microscopy allowed Niels Høiby to link chronic infections to the presence of bacterial aggregates. 11 In parallel, John William Costerton studied bacteria
in natural environments and observed the prevalence of bacteria attached to surfaces (called sessile bacteria) compared to free-floating (planktonic) bacteria. 12 In 1978, his pioneering work
led to the hypothesis that sessile bacteria exhibit a different phenotype than planktonic bacteria. When adhering to a surface, bacteria grow as a community structured by a network of fibers, called glycocalyx. 13 The term glycocalyx was later replaced by the word biofilm, which
is the term used nowadays to define an aggregate of microorganisms embedded in a self-produced matrix of extracellular polymeric substances that are adherent to each other and/or a surface. 14,15
As illustrated in Figure 2, biofilm development follows several steps: 1. attachment of planktonic cells to a surface with a conditioning layer; 2. irreversible attachment through extracellular polymeric substances (matrix); 3. microcolony formation through cell aggregation and matrix synthesis; 4. biofilm maturation leading to three-dimensional architectures; and 5. biofilm cells dispersal. 16 The matrix accounts for the majority of the biomass of the biofilm and
is composed of polysaccharides, proteins, extracellular DNA, and lipids. 17
Biofilms are widely recognized as a major threat to human health, being responsible for most chronic infections, contributing to pathogenesis of infections and increasing bacterial survival.
14,18 As biofilm cells are estimated to survive up to 1000 times higher concentrations of
antibiotics in comparison to planktonic cells, the treatment of biofilm-related infections is extremely challenging. 19 There are several reasons why biofilm cells are much more difficult
to kill than planktonic cells. Biofilms provide homeostasis and protection from environmental stresses, such as immune system and antimicrobial agents, to bacterial cells. 19,20 In addition,
the viscoelastic nature of biofilms confers resistance to mechanical challenges and limits removal from surfaces. 21
5 Biofilm formation
Figure 2. The top figure shows one of the first transmission electron micrograph of bacteria attached by their glycocalyx (later understood to be extracellular polymeric substances). 13 The schematic at the bottom represents the currently accepted model of biofilm development: initial attachment, irreversible attachment, early development of biofilm architecture, maturation, dispersion. 16
Pseudomonas aeruginosa
Among the most dangerous AMR bacterial strains for human health, Pseudomonas aeruginosa a Gram-negative bacterium belongs to the class of gamma-proteobacteria and is responsible for acute and chronic infections in humans. 22 Genomes of P. aeruginosa strains
consist of one circular chromosome with a size varying between 5.5 and 7.0 Mb, larger than most sequenced bacteria. 23 Complete sequencing of P. aeruginosa PAO1 reveals 5,570 open
reading frames, showing a greater functional diversity than its close relative, Escherichia coli.
24 Such a large genetic repertoire confers high metabolic versatility and thus the ability to utilize
various sources of nutrients and to adapt to most environments. 22
All these characteristics are making P. aeruginosa infections difficult to treat and the prevalence of multidrug resistant P. aeruginosa strains has increased from 1% in 1994 to 16% in 2002. 25 Increasing the mortality rate, multidrug resistant P. aeruginosa is also associated
with a significant economic burden, which is estimated to increase the hospital cost per admission by more than 3 fold relative to non-resistant strains. 26 Scientists have raised
awareness towards P. aeruginosa by regrouping it in the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group, alongside the most dangerous pathogens in modern society. 27 Additionally, the World Health Organization has categorized
carbapenem-resistant P. aeruginosa as a critical priority for the development of new treatments. 28
Clinical relevance of P. aeruginosa
Along with its ability to colonize most environments, P. aeruginosa is an opportunistic pathogen responsible for many nosocomial and material-related infections for humans. Hospital-acquired infections are a major threat to human health and constitute one of the leading causes of deaths in Europe and the United States. 29 Among them, P. aeruginosa is estimated to
account for 7% of all healthcare-associated infections in the United States each year. 30 These
infections often occur through invasive medical devices or surgical procedures, leading to ventilator-associated pneumonia and bloodstream and urinary tract infections. 29 Urinary tract
infections are a common type of infections affecting 150 million people each year worldwide and, if left untreated, lead to bacteremia and host tissue damage through toxin production. 31
Mortality and morbidity associated with P. aeruginosa are high, up to 13% for ventilator-associated pneumonia, and vary according to the patient health and the stage of the disease.
32 Additionally, P. aeruginosa is one of the main actors responsible for the mortality of patients
suffering from cystic fibrosis conditions. 33 Cystic fibrosis is an autosomal recessive disease
resulting in abnormal ion transport in airway epithelial cells. Such a defect leads to the accumulation of mucus in lungs and compromises airway defenses and inflammatory cell
7 Mode of action of P. aeruginosa generating AMR
function. 34 With an incidence of around 1 in 3,000 live births in northern Europe, infants with
cystic fibrosis are more likely to be infected by P. aeruginosa, which persists until death in almost 70% of the adults. 35 For therapy, administration of an anti-pseudomonal β-lactam
associated with aminoglycosides or fluoroquinolones is recommended but the clinical outcome is dependent on the emergence of resistance. 36
Mode of action of P. aeruginosa generating AMR
Bacterial survival to high doses of antibiotics is commonly referred to as resistance. However, it is known that there are three different ways leading to bacterial survival, that also apply to P. aeruginosa. In the proper sense of the term, resistance refers to the ability of bacteria to grow in the presence of antibiotics, while tolerance is the ability to survive transient antibiotic treatment, and persistence is the ability of a subclonal population to survive. 37 Additionally,
the mechanisms allowing bacteria to escape antibiotic treatment can be intrinsic, acquired and adaptive. 38 Intrinsic mechanisms correspond to the inherent characteristics of P. aeruginosa,
such as restricted membrane permeability, efflux system pumping antibiotics out of the cells, and antibiotic inactivating enzymes. 39 Acquired mechanisms lead to survival through
mutations in chromosomal genes or acquisition of resistance genes via horizontal gene transfer. 40 Finally, P. aeruginosa can respond to environmental stimuli by reversibly adapting
its transcriptome and proteome in order to survive. 41 Upon exposition to gentamicin-loaded
bone cement, P. aeruginosa has been reported to enhance matrix production leading to decreased susceptibility to antibiotics and thus failure of implementation. 42 The formation of
biofilms constitutes a major mechanism of adaptation, conferring tolerance to antibiotics through inhibition of antimicrobial penetration, physiological heterogeneities of bacterial cells, biofilm-specific stress responses, specific gene expressions, intercellular interactions, and presence of persister cells. 19,43 Biofilms also contribute in AMR spreading within its community
by promoting horizontal gene transfer. Using an in vivo model of infection, it has been demonstrated that P. aeruginosa in biofilms exhibits extreme tolerance to antibiotics and that the concentration required to achieve complete biofilm eradication is impossible to be applied in lungs. 44
Knowledge gap
The development of new antimicrobial agents is a necessity to control infections caused by P. aeruginosa biofilms. However, the task is far from easy and establishing a sustainable antibiotic discovery platform is considered one of the greatest challenges in the 21st century. 45 Current antibiotic screening is based on target-directed strategies and one of the major
bottlenecks is the identification of a suitable target. The perfect target would be a conserved Mode of action of P. aeruginosa generating AMR
and essential function, enzyme or structure in bacteria not prone to rapid resistance development, not sharing similarities to those from the mammalian host, and possessing a potential for druggability and efficient pharmacological properties. 46 Following these criteria,
the latest new class of antibiotics against Gram-negative bacteria was quinolone, released over 50 years ago. 45 Nowadays, most antibiotics in clinical trials are derivatives of known
classes of antibiotics, which provide little help against bacteria already resistant to the original antibiotics. 6 Thus, new targets need to be identified. To this end, it is necessary to understand
how bacterial cells generate resistance, especially in the biofilm mode of growth.
Aim of the thesis
P. aeruginosa is a pathogen of high clinical relevance, showing multiple mechanisms of adaptation and high recalcitrance to our current antimicrobial agents. Over the past decades, AMR bacteria are becoming more and more frequent and can sometimes lead to untreatable infections. In these dramatic cases, patients and doctors have little options, which can lead to amputations or use of experimental treatments. Thus, new therapeutic treatments and strategies need to be developed.
Founded by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) and part of the partnership against Biofilm-associated Expression, Acquisition and Transmission of Antimicrobial Resistance (BEAT-AMR), this PhD project aims at identifying targets responsible for antibiotic tolerance in P. aeruginosa biofilms. Such investigations can provide a better understanding of the mechanisms employed by P. aeruginosa to overcome antibiotic treatment in the context of biofilm-related infections. Gene identification can highlight promising targets for drug development, unraveling new classes of antibiotics or improving the efficiency of existing drugs against P. aeruginosa biofilms. A focus is put on the antibiotics gentamicin, an aminoglycoside commonly used to treat P. aeruginosa infections but often rendered inefficient due to resistance, and colistin, a last resort antibiotic used to treat multidrug resistant P. aeruginosa in cystic fibrosis patients. Unraveling the link between genes and tolerance to these antibiotics can provide useful insights for clinicians regarding treatment options.
More specifically, the importance of specific genes for biofilm physiology is investigated through transposon insertion mutants of P. aeruginosa MPAO1. An in vitro screening method with high throughput is developed to assess biofilm formation and tolerance to antibiotics. In Chapter 2, a preliminary screening is performed by using a random selection of genes. In Chapter 3, a thorough screening is done by focusing on biofilm-associated genes in P. aeruginosa. In Chapter 4, a more in-depth study is performed on the P. aeruginosa mutant missing the flgE gene, identified as one key contributor for biofilm tolerance to gentamicin.
9 References
Finally, Chapter 5 discusses the overall contribution of this thesis to the struggle against P. aeruginosa biofilms and recommendations for potential future investigations are put forward.
References
1. Homann, M. et al. Microbial life and biogeochemical cycling on land 3,220 million years ago. Nat. Geosci. 11, 665–671 (2018).
2. Morens, D. M. & Fauci, A. S. Emerging pandemic diseases: how we got to COVID-19. Cell 182, 1077–1092 (2020).
3. Wiersinga, W. J., Rhodes, A., Cheng, A. C., Peacock, S. J. & Prescott, H. C. Pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (COVID-19): a review. JAMA 324, 782–793 (2020).
4. Porter, J. R. Antony van Leeuwenhoek. Tercentenary of his discovery of bacteria. Bacteriol. Rev. 40, 260–269 (1976).
5. Berche, P. Louis Pasteur, from crystals of life to vaccination. Clin. Microbiol. Infect. 18, 1–6 (2012).
6. Hutchings, M., Truman, A. & Wilkinson, B. Antibiotics: past, present and future. Curr. Opin. Microbiol. 51, 72–80 (2019).
7. Fleming, A. Penicillin. Nobel Lecture (1945).
8. O’Neill, J. Review on antimicrobial resistance: tackling a crisis for the health and wealth of nations. Rev. Antimicrob. Resist. https://amr-review.org/Publications.html (2014). 9. Ritchie, H. & Roser, M. Causes of Death. OurWorldInData.org, Available online at:
https://ourworldindata.org/ca (2018).
10. Ventola, C. L. The antibiotic resistance crisis part 1: causes and threats. P&T 40, 277– 283 (2015).
11. Høiby, N. A short history of microbial biofilms and biofilm infections. APMIS 125, 272– 275 (2017).
12. Geesey, G. G., Mutch, R., Costerton, J. W. & Green, R. B. Sessile bacteria: an important component of the microbial population in small mountain streams. Limnol. Oceanogr. 23, 1214–1223 (1978).
13. Costerton, J. W., Geesey, G. G. & Cheng, K.-J. How bacteria stick. Sci. Am. 238, 86– 95 (1978).
14. Costerton, J. W., Stewart, P. S. & Greenberg, E. P. Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322 (1999).
15. Flemming, H.-C. et al. Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol. 14, 563–575 (2016).
16. Stoodley, P., Sauer, K., Davies, D. G. & Costerton, J. W. Biofilms as complex differentiated communities. Annu. Rev. Microbiol. 56, 187–209 (2002).
17. Flemming, H. C. & Wingender, J. The biofilm matrix. Nat. Rev. Microbiol. 8, 623–633 (2010).
18. Vestby, L. K., Grønseth, T., Simm, R. & Nesse, L. L. Bacterial biofilm and its role in the pathogenesis of disease. Antibiotics 9, 59 (2020).
19. Lebeaux, D., Ghigo, J.-M. & Beloin, C. Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. Microbiol. Mol. Biol. Rev. 78, 510–543 (2014).
20. Maurice, N. M., Bedi, B. & Sadikot, R. T. Pseudomonas aeruginosa biofilms: host response and clinical implications in lung infections. Am. J. Respir. Cell Mol. Biol. 58, 428–439 (2018).
21. Peterson, B. W. et al. Viscoelasticity of biofilms and their recalcitrance to mechanical and chemical challenges. FEMS Microbiol. Rev. 39, 234–245 (2015).
22. Moradali, M. F., Ghods, S. & Rehm, B. H. A. Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence. Front. Cell. Infect. Microbiol. 7, 39 (2017).
23. Klockgether, J., Cramer, N., Wiehlmann, L., Davenport, C. F. & Tümmler, B. Pseudomonas aeruginosa genomic structure and diversity. Front. Microbiol. 2, 150 (2011).
24. Stover, C. K. et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964 (2000).
25. D’Agata, E. M. C. Rapidly rising prevalence of nosocomial multidrug-resistant, Gram-negative bacilli a 9-year surveillance study. Infect. Control. Hosp. Epidemiol. 25, 842– 846 (2004).
26. Morales, E. et al. Hospital costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition. BMC Health Serv. Res. 12, 122 (2012).
27. Boucher, H. W. et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 48, 1–12 (2009).
28. Tacconelli, E. et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 18, 318– 327 (2018).
29. Peleg, A. Y. & Hooper, D. C. Hospital-acquired infections due to Gram-negative bacteria. N. Engl. J. Med. 362, 1804–1813 (2010).
30. Weiner, L. M. et al. Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the national healthcare safety network at the centers for disease control and prevention, 2011-2014. Infect. Control Hosp. Epidemiol. 37, 1288–1301 (2016).
31. Flores-Mireles, A. L., Walker, J. N., Caparon, M. & Hultgren, S. J. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat. Rev. Microbiol. 13, 269–284 (2015).
32. Melsen, W. G. et al. Attributable mortality of ventilator-associated pneumonia: a meta-analysis of individual patient data from randomised prevention studies. Lancet Infect. Dis. 13, 665–671 (2013).
33. Høiby, N., Ciofu, O. & Bjarnsholt, T. Pseudomonas aeruginosa biofilms in cystic fibrosis. Future Microbiol. 5, 1663–1674 (2010).
34. Ratjen, F. et al. Cystic fibrosis. Nat. Rev. Dis. Prim. 1, 15010 (2015).
35. Döring, G. et al. Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus. Eur. Respir. J. 16, 749–767 (2000).
36. Bassetti, M., Vena, A., Croxatto, A., Righi, E. & Guery, B. How to manage Pseudomonas aeruginosa infections. Drugs Context 7, 1–18 (2018).
11 References
37. Brauner, A., Fridman, O., Gefen, O. & Balaban, N. Q. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat. Rev. Microbiol. 14, 320–330 (2016).
38. Pang, Z., Raudonis, R., Glick, B. R., Lin, T. J. & Cheng, Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol. Adv. 37, 177–192 (2019).
39. Blair, J. M. A., Webber, M. A., Baylay, A. J., Ogbolu, D. O. & Piddock, L. J. V. Molecular mechanisms of antibiotic resistance. Nat. Rev. Microbiol. 13, 42–51 (2015).
40. Partridge, S. R., Kwong, S. M., Firth, N. & Jensen, S. O. Mobile genetic elements associated with antimicrobial resistance. Clin. Microbiol. Rev. 31, e00088-17 (2018). 41. Sandoval-Motta, S. & Aldana, M. Adaptive resistance to antibiotics in bacteria: a
systems biology perspective. Wiley Interdiscip. Rev. Syst. Biol. Med. 8, 253–267 (2016). 42. Neut, D., Hendriks, J. G. E., van Horn, J. R., van der Mei, H. C. & Busscher, H. J. Pseudomonas aeruginosa biofilm formation and slime excretion on antibiotic-loaded bone cement. Acta Orthop. Scand. 76, 109–114 (2005).
43. Hall, C. W. & Mah, T. F. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiol. Rev. 41, 276–301 (2017). 44. Wang, H., Wu, H., Ciofu, O., Song, Z. & Høiby, N. In vivo
pharmacokinetics/pharmacodynamics of colistin and imipenem in Pseudomonas aeruginosa biofilm infection. Antimicrob. Agents Chemother. 56, 2683–2690 (2012). 45. Hoffman, P. S. Antibacterial discovery: 21st century challenges. Antibiotics 9, 213
(2020).
46. Silver, L. L. Challenges of antibacterial discovery. Clin. Microbiol. Rev. 24, 71–109 (2011).
