Pseudomonas aeruginosa biofilms and their partners in crime

1 Pseudomonas aeruginosa biofilms and their partners in crime 1 2 Maria del Mar Cendra1* and Eduard Torrents1,2* 3 4 5 1Bacterial infections and antimicrobial therapies group. Institute for Bioengineering of 6 Catalonia (IBEC), The Barcelona Institute of Science and Technology; Baldiri Reixac 15-21, 7 08028, Barcelona, Spain. 2Microbiology Section, Department of Genetics, Microbiology and 8 Statistics, Faculty of Biology, University of Barcelona, 643 Diagonal Ave., 08028, Barcelona, 9 Spain. 10 11 12 *Corresponding author: 13 Dr. Eduard Torrents: [email protected] 14 Dr. Maria del Mar Cendra: [email protected] 15 16 Keywords: Pseudomonas aeruginosa, biofilms, polymicrobial, chronic infections, P. 17 aeruginosa models, antimicrobials 18 19 20 Manuscript - Clear Version of the revised manuscript Click here to view linked References 2 Abstract 21 Pseudomonas aeruginosa biofilms and the capacity of the bacterium to coexist and 22 interact with a broad range of microorganisms have a substantial clinical impact. This review 23 focuses on the main traits of P. aeruginosa biofilms, such as the structural composition and 24 regulatory networks involved, placing particular emphasis on the clinical challenges they 25 represent in terms of antimicrobial susceptibility and biofilm infection clearance. Furthermore, 26 the ability of P. aeruginosa to grow together with other microorganisms is a significant 27 pathogenic attribute with clinical relevance; hence, the main microbial interactions of 28 Pseudomonas are especially highlighted and detailed throughout this review. This article also 29 explores the infections caused by single and polymicrobial biofilms of P. aeruginosa and the 30 current models used to recreate them under laboratory conditions. Finally, the antimicrobial and 31 antibiofilm strategies developed against P. aeruginosa mono and multispecies biofilms are 32 detailed at the end of this review. 33 34 3 1. Introduction 35 The ability of Pseudomonas aeruginosa to colonize medical devices and human tissues 36 while growing in resistant communities called biofilms is a worldwide public health concern. 37 Biofilms are bacterial communities that grow together embedded in an extracellular matrix 38 (ECM), which is a fundamental structural component of the bacterial community and acts as a 39 protective shield (Ma et al., 2009). Bacteria modulate their gene expression during adaptation 40 to biofilm growth, promoting phenotypically opposite behavior compared to their planktonic 41 counterparts. Bacterial communication via the quorum sensing (QS) network plays a critical 42 role during biofilm establishment, namely, in regulating the genes involved in biofilm 43 development (Schuster and Greenberg, 2006). P. aeruginosa biofilms have increased antibiotic 44 tolerance and are more resistant to host responses than their planktonic counterparts, which 45 makes the clearance of these biofilms difficult and infections chronic (Lebeaux et al., 2014; 46 Maurice et al., 2018). 47 A critical clinical trait of P. aeruginosa is its capacity to interact and coexist with other 48 microorganisms in multispecies communities. From a clinical point of view, these interactions 49 are usually detrimental to the patient, as infections caused by multiple species are often 50 associated with worse prognosis (Peters et al., 2012). On the other hand, from a biotechnological 51 perspective, there is a challenge to recreate the optimal conditions to grow multiple bacterial 52 species simultaneously. P. aeruginosa can interact with other bacteria, fungi and viruses and 53 together infect a wide range of human tissues (Filkins et al., 2015; Hendricks et al., 2016; Smith 54 et al., 2015). Due to the clinical challenge of P. aeruginosa biofilms and the recalcitrant 55 infections they cause, science has moved toward developing efficient and alternative 56 antimicrobial strategies to clear P. aeruginosa biofilms (Barraud et al., 2009; Guillon et al., 57 2018; Ibaraki et al., 2020; Mwangi et al., 2019). 58 4 This review outlines the main aspects of P. aeruginosa biofilms and the clinical burden 59 they represent; describes P. aeruginosa infections, importantly focusing on the polymicrobial 60 interactions of this bacterium and the relative clinical outcomes; and finally, it discusses 61 the current models used to recreate P. aeruginosa polymicrobial biofilms under laboratory 62 conditions and the antimicrobial therapeutics used against P. aeruginosa biofilms. 63 64 5 2. Extracellular matrix of Pseudomonas aeruginosa biofilms: the structural 65 basis of the clinical threat. 66 The ECM is the body of the biofilm. It maintains biofilm integrity by holding the 67 bacterial community together and confers biofilm protection against antimicrobials and the host 68 immune response. Hence, the ECM is a fundamental component of this multifactorial structure 69 and is composed mainly of a mix of exopolysaccharides (EPS), extracellular DNA (eDNA) and 70 proteins (Ma et al., 2009). 71 Psl and Pel are the two main polysaccharides present in the ECM of P. aeruginosa strains 72 and are crucial for the integrity of the biofilm. Psl is a neutral pentasaccharide composed of D- 73 glucose, D-mannose and L-rhamnose, while Pel is a cationic exopolysaccharide comprising 1- 74 4 linked galactosamine and glucosamine sugars (Billings et al., 2013; Franklin et al., 2011; 75 Jennings et al., 2015). However, during an in vivo infection, mutations in the anti-sigma factor 76 encoding the mucA gene result in overproduction of the alginate polysaccharide and change the 77 architecture of the biofilm’s ECM (Martin et al 1993). Alginate is an anionic polymer composed 78 of -D-mannuronic acid and -L-guluronic acid. Biofilms that contain bacteria overproducing 79 alginate occupy more space than nonmucoid biofilms, which are more densely packed (Ma et 80 al., 2012). Each polysaccharide (Psl, Pel and alginate) provides different physiological 81 properties to the biofilm matrix, and it is associated with different stages of biofilm 82 development. Even though P. aeruginosa has the capacity to produce the three types of 83 polysaccharide, it only does one type at any given time (Franklin et al., 2011). 84 The initiation and maintenance of biofilms are promoted by the interaction of Psl with 85 the matrix adhesin CdrA. Within the matrix, bound CdrA-Psl forms robust and protease- 86 resistant bacterial aggregates that fortify the biofilm structure (Borlee et al., 2010; Ma et al., 87 2009; Reichhardt et al., 2018). CdrA can also attach other yet-unknown EPS, contributing to 88 biofilm formation and stabilizing the structure (Reichhardt et al., 2018). CdrA, Psl and Pel are 89 6 bis-(3′-5′)-cyclic diguanosine monophosphate (c-di-GMP)-dependent molecules (Borlee et al., 90 2010). c-di-GMP is a secondary messenger that transduces the environmental signal into 91 different cellular processes (Jenal and Malone, 2006). Under low concentrations of c-di-GMP, 92 P. aeruginosa biofilms disperse and become planktonic cells. High levels of cAMP have been 93 directly related to low c-di-GMP content, thus contributing to the inhibition of P. aeruginosa 94 biofilm formation (Almblad et al., 2019). Alginate is regulated by c-di-GMP at the post- 95 translational level (Whitney et al., 2015). Additionally, P. aeruginosa produces two soluble 96 lectins: LecA and LecB. The binding between LecB and mannose present in Psl favors the 97 positioning of Psl within the matrix, enhancing bacterial retention and aggregation (Passos da 98 Silva et al., 2019). LecA shows specificity for binding galactose, N-acetyl-d-galactosamine and 99 glucose, and it contributes to biofilm formation by cross-linking with these residues present in 100 the biofilm matrix as well as with other glycoproteins and bacterial polysaccharides (e.g., LPS) 101 (Diggle et al., 2006; Mitchell et al., 2005; Sabin et al., 2006). 102 A critical and clinically relevant event occurs in vivo, during the establishment of P. 103 aeruginosa infection, with the organism becoming mucoid and overproducing alginate 104 polysaccharide. From that moment on, P. aeruginosa induces the transition from an intermittent 105 to chronic infection (Martin et al., 1993). Mucoid strains generally produce less Psl than 106 nonmucoid P. aeruginosa strains. Nevertheless, the interactions between this EPS and the 107 components of the matrix are also required to keep bacteria together and form robust and mature 108 biofilms (Jones and Wozniak, 2017; Ma et al., 2012). 109 All three polysaccharides (Psl, Pel, alginate) play a role in biofilm adhesion, scaffolding, 110 and stability. However, they differ in terms of biofilm protection. While Psl confers protection 111 against the immune cells, Pel is shown to defense the biofilm against antimicrobial treatment. 112 On the other hand, alginate production protects from both antimicrobials and the host immune 113 7 response and confers additional protection to hostile environments as, for instance, the oxidative 114 stress created during phagocytosis (Karygianni et al., 2020). 115 eDNA is a major component of the P. aeruginosa biofilm matrix that changes depending 116 on the biofilm maturity and the surrounding environment (Whitchurch et al., 2002). In P. 117 aeruginosa, eDNA is produced through a process of explosive cell lysis that subsequently forms 118 membrane vesicles by engulfing DNA and other cytosolic content. eDNA is then released in a 119 mechanism that is regulated by both QS-dependent and QS-independent mechanisms (Turnbull 120 et al., 2016). Once released, eDNA interacts with extracellular Ca2+ and, via “cationic bridging”, 121 induces bacterial aggregation, promoting biofilm formation and subsequent maturation. 122 Therefore, eDNA is required for the initial establishment of the biofilm (Das et al., 2014; 123 Whitchurch et al., 2002). eDNA has been detected to interact with Psl (Wang et al., 2015) and 124 Pel (Jennings et al., 2015) polysaccharides. During an infection, the host also impacts the 125 presence of eDNA in the biofilm matrix through interactions with immune cells during the 126 inflammatory response. While eDNA has been shown inside biofilms in in vitro studies, in in 127 vivo biofilms, it has been shown to be concentrated in the external part of the biofilms (Alhede 128 et al., 2020; Ciszek-Lenda et al., 2019; Whitchurch et al., 2002). P. aeruginosa strains, namely, 129 the “rugose small colony variants” (RSCVs), isolated from patients with chronic infections are 130 hyperbiofilm-forming strains that, unlike common laboratory strains, have fragmented eDNA 131 within the matrix that leads to a more resistant structure (Deng et al., 2020). RSCVs are 132 associated with high levels of c-di-GMP (Malone et al., 2010), in addition, to be able to produce 133 alginate and Psl polysaccharides simultaneously (Franklin et al., 2011). 134 However, eDNA is more than just a structural biofilm component; it also influences the 135 transcriptome of P. aeruginosa. This anionic polymer modulates the expression of antibiotic 136 resistance genes such as ß-lactamases and aminoglycoside resistance genes as well as the 137 expression of multidrug efflux pumps (e.g., EmrAB). Additionally, it alters metal homeostasis 138 8 by chelating cationic ions and controlling the expression of different metal uptake and efflux 139 systems. The acidification of the biofilm confers tolerance to acidic environments, making 140 biofilms able to resist the infection site or phagocytose acidified vacuoles. Importantly, eDNA 141 traps nutrients in addition to inducing genes to be able to use DNA as a nutrient source for 142 phosphate, nitrogen or carbon (Lewenza et al., 2020; Mulcahy et al., 2008; Wilton et al., 2016). 143 Altogether, eDNA promotes all the hallmark features of biofilms. A recently published mini- 144 review addresses the different aspects of P. aeruginosa eDNA release and interactions very 145 accurately (Sarkar, 2020). 146 147 3. P. aeruginosa polymicrobial coexistence 148 In nature, biofilms are often polymicrobial structures, meaning that different microbial 149 species can interact and coexist within the same biofilm community. In disease, microbial 150 interactions can affect a patient’s prognosis. In this sense, the synergic interactions of organisms, 151 whereby the combined effect is more significant than that produced by individual bacteria, can 152 worsen the outcome of the patient (Murray et al., 2014). P. aeruginosa can grow and coexist 153 with a wide range of microorganisms, including bacteria, fungi and viruses (Figure 1). 154 155 3.1. P. aeruginosa with Staphylococcus aureus 156 One of the major partners in crime of P. aeruginosa is Staphylococcus aureus. This 157 partnership can cause severe chronic infections promoted by biofilm aggregates in infected 158 wounds and lungs, especially in diabetic foot ulcers and cystic fibrosis (CF). The presence of S. 159 aureus and the Pel production of P. aeruginosa have been shown to increase the surface 160 coverage and microcolony size of biofilms formed by both P. aeruginosa and S. aureus, and 161 diguanylate cyclase SiaD is required for competitiveness between them (Chew et al., 2018). In 162 CF-affected lungs, P. aeruginosa takes advantage of the physiology generated by the disease 163 9 and the lack of iron present in the environment to produce 4-hydroxy-2-heptylquinoline-N-oxide 164 (HQNO). HQNO inhibits S. aureus growth by shifting its metabolism to fermentation, which 165 eventually promotes the killing of S. aureus, and P. aeruginosa can use the iron that 166 Staphylococcus stored (Filkins et al., 2015; Mashburn et al., 2005; Nguyen et al., 2015). P. 167 aeruginosa produces the endopeptidase LasA, which has also been suggested to help for 168 acquiring the iron from S. aureus (Mashburn et al., 2005). Transcriptional profiles performed in 169 early cocultures of both bacteria reflect the metabolic adaptation and competition for glutamine, 170 as nitrogen and energy sources, that the organisms face when they grow together rather than the 171 expression of host-directed virulence factors (Tognon et al., 2019). It has also been seen that 172 during P. aeruginosa coinfection with Gram-positive bacteria such as S. aureus, P. aeruginosa 173 senses the N-acetyl glucosamine (GlcNAc) of its peptidoglycan as a cue to produce multiple 174 virulent factors with lytic activity against prokaryotic and eukaryotic cells, therefore enhancing 175 host killing (Korgaonkar et al., 2013). P. aeruginosa production of the cis-2-decenoic acid 176 promotes biofilm dispersal of different bacteria, including S. aureus (Davies and Marques, 177 2009). 178 On the other hand, S. aureus can use the HQNO molecule produced by P. aeruginosa to 179 increase its tolerance to certain antibiotics, such as vancomycin or tobramycin. Prolonged 180 coculture growth of P. aeruginosa and S. aureus or increased S. aureus exposure to HQNO, 181 which is abundant in CF sputum, selects for resistant S. aureus small-colony variants (SCV), in 182 a mechanism dependent on the transcriptional factor sigma B (SigB) (Mitchell et al., 2010). 183 Furthermore, P. aeruginosa influences S. aureus susceptibility to antibiotics such as 184 vancomycin, gentamycin or ciprofloxacin (Biswas et al., 2009; Cendra et al., 2019; Hoffman et 185 al., 2006; Orazi and O'Toole, 2017). Recent findings indicate that S. aureus extracellular 186 metabolites (e.g., adhesins, enzymes, polysaccharides and peptides) can suppress P. aeruginosa 187 growth, which leads to an increase in P. aeruginosa susceptibility to antibiotics such as 188 16 composition of the ECM and the mechanisms by which the QS modulates the ECM 338 development. 339 340 4.2. Effect of QS on infection and P. aeruginosa polymicrobial interactions 341 The QS of P. aeruginosa significantly impacts on the polymicrobial interactions 342 promoted by the bacterium. Using a wound-like medium, it was demonstrated that the QS of P. 343 aeruginosa is inhibited by the albumin present in the serum in vitro, thus making the bacteria 344 unable to produce the virulence factors that kill S. aureus and allowing the survival of the cocci 345 in the presence of P. aeruginosa (Smith et al., 2017). It has also been seen that alginate protects 346 S. aureus from killing by P. aeruginosa when both organisms are growing in coculture due to a 347 downregulation of the pvdA gene, which is required to produce the siderophore pyoverdine and 348 the QS system Pseudomonas Quinolone System (PQS) in P. aeruginosa (Price et al., 2020). The 349 PQS is responsible for producing the phenazine pyocyanin, which is induced by the presence of 350 S. aureus (among other Gram-positive species and conditions), inhibiting the oxidative 351 respiration of the cocci while promoting the selection of SCV (Biswas et al., 2009). 352 Additionally, a recent study revealed that P. aeruginosa suppresses its antimicrobial activity 353 against S. aureus through a mechanism that involves Pseudomonas AAA+ ClpXP protease 354 activity on critical proteins needed to produce PQS and C4-HSL QS signal molecules (Yang et 355 al., 2020). Otherwise, when P. aeruginosa interacts with C. albicans, pyocyanin secretion by P. 356 aeruginosa is toxic in C. albicans, which counterattacks by producing the QS molecule farnesol. 357 Farnesol downregulates the transcription of the pyocyanin mediator gene pqsA (Cugini et al., 358 2007). During the colonization of the CF lung, farnesol may also have a protective role by 359 reducing the levels of pyocyanin (Peters et al., 2012). In the interaction between P. aeruginosa 360 and S. maltophilia, the latter produces a diffusible signal factor that influences the structure of 361 P. aeruginosa biofilms (Ryan et al., 2008). Furthermore, S. maltophilia produces the fatty acid 362 17 cis-9-octadecenoic that quenches the AHL signal of P. aeruginosa, inhibiting its biofilm 363 formation (Singh et al., 2013). 364 Additionally, mutants of the QS regulator LasR have been associated with lung disease 365 progression in CF-affected people (Hoffman et al., 2009). In these lasR mutant strains, QS 366 remains active by regulating RhlR in a mechanism independent of LasR (Chen, R. et al., 2019). 367 In a study that used a CF isolate, RhlR was recognized as critical for establishing chronic 368 infection and the generation of cell toxicity in a 3D lung epithelium aggregated model (Cruz et 369 al., 2020). 370 371 5. Clinical implications of P. aeruginosa biofilms – a public health issue 372 Microbial biofilms are involved in 65% of infectious diseases and more than 80% of 373 chronic infections. P. aeruginosa is a leading nosocomial pathogen associated with this type of 374 healthcare infection, and it is almost never found infecting alone (Bisht et al., 2020). The nature 375 of P. aeruginosa biofilms per se is a challenge to the current known antimicrobial treatments 376 and a nightmare for physicians. 377 378 5.1 The antimicrobial challenge of P. aeruginosa biofilms 379 The primary shield of P. aeruginosa biofilms is its ECM. The chemistry of the ECM 380 hinders the penetration of positively charged antibiotics, e.g., aminoglycosides, which are 381 sequestered by their components and impede their diffusion (Wilton et al., 2016). Biofilm 382 growth confers intrinsic antimicrobial tolerances, sometimes requiring more than 1000 times 383 the dose of antibiotic to be cleared than is needed to treat planktonic bacteria. Furthermore, the 384 clinically derived mucoid phenotype, with alginate overproduction in the ECM, is even more 385 antibiotic tolerant than the parental P. aeruginosa. Altogether, the high antibiotic concentrations 386 needed to treat these biofilms are hard to achieve inside the host without causing toxicity 387 18 (Goltermann and Tolker-Nielsen, 2017; Hengzhuang et al., 2013; Macia et al., 2014). 388 Additionally, the P. aeruginosa biofilm community is constituted by different subpopulations 389 of microorganisms, which have been adapted to the different microenvironments present in the 390 multicellular system. Therefore, the bacteria residing in the inner parts of the biofilms display 391 low metabolic activity due to the lack of oxygen and nutrients that are essentially consumed by 392 the cells growing at the biofilm periphery, which consequently inactivates major antibiotic 393 targets. For example, low DNA and protein synthesis affect quinolone and aminoglycoside 394 bactericidal effects, respectively (Ciofu and Tolker-Nielsen, 2019; Stewart et al., 2016). This 395 low-metabolic Pseudomonas can lead to the formation of persister cells, which are bacteria less 396 susceptible to antibiotics and responsible for causing reinfections (Lewis, 2010). Furthermore, 397 the hypoxic environment present in the inner parts of the biofilm is another tolerance mechanism 398 attributed to P. aeruginosa biofilms since the lack of oxygen impedes the ROS necessary for 399 the bactericidal effect of some antibiotics, such as tobramycin or ciprofloxacin (Borriello et al., 400 2004; Van Acker and Coenye, 2017). Antibiotic tolerance is also attributed to genetic changes 401 occurring in these P. aeruginosa subpopulations, where the activation of multidrug efflux 402 pumps due to mechanisms led by the high levels of cyclic di-GMP present in the biofilm pump 403 antibiotics such as ciprofloxacin, gentamycin and tobramycin out of the bacterial cell at rates 404 ten times higher than those in planktonic cells (Gupta et al., 2014; Poudyal and Sauer, 2018). 405 Additionally, these transcriptomic changes in biofilm-forming Pseudomonas also occur in many 406 other genes that are absent in free-living Pseudomonas that contribute to specific antimicrobial 407 tolerance (Ciofu and Tolker-Nielsen, 2019). 408 Spontaneous mutations accompany the increased capacity of antimicrobial tolerance and 409 confer resistance to certain antibiotics in Pseudomonas due to antimicrobial pressure. In 410 addition to antibiotic resistance, the heterogeneity of the biofilm community and the different 411 stresses and pressures on the subpopulations of the bacteria induce differential spontaneous 412 19 mutations that benefit biofilm adaptability and persistence (Bjedov et al., 2003; Ciofu and 413 Tolker-Nielsen, 2019; Perron et al., 2007). In this sense, RSCV in CF has increased resistance 414 to antibiotics, and their persistence in the CF lung is thought to be due to the emergence of 415 multidrug-resistant (MDR) variants of the mucoid phenotype (Ciofu et al., 2015; Drenkard and 416 Ausubel, 2002). 417 418 5.2 The battle of the host immune response to clear P. aeruginosa biofilms 419 In response to a chronic infection generated by P. aeruginosa biofilm, the host responds 420 by attacking the bacterial community with different types of immune cells from both the innate 421 and adaptive systems (Maurice et al., 2018). There is an exacerbation of inflammation led by 422 the presence of polymorphonuclear leukocytes (PMNs). Although PMNs have been detected 423 surrounding P. aeruginosa aggregates, they are unable to penetrate biofilm structures and 424 therefore eradicate them (Bjarnsholt et al., 2009). PMNs are responsible for neutrophil 425 extracellular trap (NET) formation in a process called NETosis. NETs are DNA lattices 426 enmeshed with PMN granule proteins secreted by PMNs. The creation of NETs has been 427 proposed as a function of neutrophils as well as other immune cells that are still capable of 428 phagocytosis and chemotaxis (Goldmann and Medina, 2012; Yipp et al., 2012). NETs aim to 429 trap bacteria and kill them with antimicrobial proteins released from neutrophil azurophilic 430 granules (Brinkmann et al., 2004). 431 Even though NET formation is an efficient antimicrobial mechanism, depending on the 432 case, P. aeruginosa has the potential to overcome it. Recently, P. aeruginosa strains that lack 433 the LasR regulator (commonly found in CF patients) have been shown to fail to promote the 434 generation of NETosis (Skopelja-Gardner et al., 2019). Furthermore, P. aeruginosa can cause 435 tolerance to NETs by changing the negative charge of the outer surface either through the 436 20 addition of aminoarabinose to lipid A in LPS or by producing surface spermidine (Halverson et 437 al., 2015; Johnson et al., 2012). 438 Importantly, under healthy conditions, humans have effective immune mechanisms to 439 clear P. aeruginosa infection. For instance, mucociliary clearance in the upper respiratory 440 system and the expression of lactoferrin have been shown to effectively block the attachment 441 and microcolony formation of P. aeruginosa, thus preventing the establishment of P. 442 aeruginosa infection (Crabbe et al., 2014; Singh et al., 2002). However, if P. aeruginosa can 443 penetrate and colonize patients affected by CF or another condition that compromises the 444 immune system, then the situation can become dire. In CF-affected people, oxygen consumption 445 caused by PMN activity inhibits proper production of the metabolic burst to kill phagocytosed 446 bacteria (Bjarnsholt et al., 2009). Furthermore, the overproduction of alginate present in the 447 aggregates of CF isolates confers additional phagocytosis protection to the biofilm (Bayer et al., 448 1991). Continuous exposure to the antigen indicates that IgG avidity against alginate does not 449 significantly increase along with the progression of chronic infection, making it difficult for the 450 immune reaction to clear the infection in CF patients (Mauch et al., 2018). Increasing the 451 problem is the capacity of clinical isolates of P. aeruginosa to induce the formation of biofilms 452 in the airways by upregulating EPS production through the assimilation of the host-derived 453 immunometabolite itaconate (Riquelme et al., 2020). 454 455 5.3 P. aeruginosa chronic infections due to biofilm formation 456 The ability of microbial biofilms to resist exposure to a high concentration of 457 antimicrobials and components of the host immune system makes Pseudomonas biofilms 458 incredibly challenging to eradicate and a public health concern (Hoiby et al., 2015). The 459 following section will focus on the most common chronic infections caused by P. aeruginosa 460 biofilms and the organism’s interactions with other microbes. Figure 4 summarizes the 461 21 characteristics, effects, and consequences of biofilm growth among the different infections. In 462 the figure are present similarities and differences between infections. 463 464 5.3.1 Cystic fibrosis 465 People often associate P. aeruginosa infections with CF disease. The pathophysiology 466 of this disease, with increased viscosity and mucus secretions which, together with the impaired 467 mucociliary function that these patients suffer, creates a perfect environment in the airways for 468 chronic microbial colonization, is the primary cause of morbidity and mortality of this disease 469 (Rajan and Saiman, 2002). P. aeruginosa plays a leading role in CF, accounting for 40-60% of 470 infections of this disease (CysticFibrosisFoundation, 2019). The microaerophilic environment 471 in the CF lungs, with clear oxygen gradients promoted by abundant mucus deposition in this 472 organ, promotes P. aeruginosa survival and subsequent growth. In this sense, we have recently 473 shown a change in the metabolism of P. aeruginosa during the transition from aerobic to 474 microaerophilic and, subsequent, anaerobic growth conditions (Pedraz et al., 2019). In CF in 475 vivo, P. aeruginosa aggregates in clusters rather than in compact biofilms and grows immersed 476 within a self-produced alginate (Lam et al., 1980; Moreau-Marquis et al., 2008; Worlitzsch et 477 al., 2002). P. aeruginosa can undergo mutations that increase its persistence in CF-affected 478 lungs. As mentioned above, the mucoid phenotype of Pseudomonas is frequently isolated from 479 CF exacerbations. Additionally, during P. aeruginosa CF infections, microorganisms can also 480 lose the flagellum and, consequently, swimming motility. Flagellar mutants are linked to the 481 RSCV phenotype, which has been recognized to overproduce Psl and Pel exopolysaccharides, 482 thus enhancing the bacterial capacity to form biofilms and persist in CF lungs (Harrison et al., 483 2020). 484 However, after many investigations involving culture-dependent and culture- 485 independent microbiological techniques as well as RNA-based studies, CF infections have been 486 22 determined to be polymicrobial and different between CF patients (Acosta et al., 2020; Filkins 487 and O'Toole, 2015). The polymicrobial community that colonizes the CF lung harbors 488 facultative as well as obligate anaerobic bacteria (Filkins and O'Toole, 2015). A recent study 489 using bioorthogonal noncanonical amino acid tagging revealed extensive heterogeneity of 490 translational activity among the CF microbiota, which is unique in every CF individual. 491 Therefore, in addition to canonical lung pathogens such as P. aeruginosa, there is a low 492 abundance of other members whose activity dynamics are determinants of the acute 493 inflammation occurring in CF, by either impacting the host or through the modulation of the 494 other pathogen’s growth and virulence (Valentini et al., 2020). Furthermore, the existence of 495 clonal strains of P. aeruginosa, a consequence of patient-patient infections, which are prevalent 496 in clusters of people who live in a defined geographical area, also impacts the microbiota of CF 497 (Parkins et al., 2018). In this direction, we have recently demonstrated that reference strains and 498 clinical isolates of P. aeruginosa behave differently during their adaptability and intracellular 499 survival into the lung epithelium. Our work shows the importance of choosing appropriate 500 strains when studying infectious processes with relevant translational outcomes (Cendra and 501 Torrents, 2020). 502 Within the polymicrobial nature of CF infection, P. aeruginosa and Staphylococcus 503 aureus have long been recognized as primary CF pathogens, with an increasing rate of MDR 504 appearance (Rutter et al., 2017). Although it is commonly assumed that S. aureus is an early 505 colonizer of CF lungs that is replaced by P. aeruginosa, a recent longitudinal retrospective, 506 single-center cohort study that included 337 patients with CF determined that P. aeruginosa 507 does not replace the S. aureus; rather, both species accumulate over time (Fischer et al., 2020). 508 509 5.3.2. Chronic wound infection 510 23 The skin, despite its role as a protective barrier, is susceptible to many infections, 511 especially when it is affected by burns, wounds or ulcers (Schittek, 2011). It is estimated that 512 approximately 1-2% of the population in developed countries will suffer from chronic wounds 513 during their lifetimes (Gottrup, 2004). P. aeruginosa, together with S. aureus, are common 514 bacteria usually involved in infections of skin injuries. Biofilms of P. aeruginosa are typically 515 associated with deep chronic wounds, which makes treatment with topical therapies difficult 516 (Fazli et al., 2009; Rabin et al., 2015). The chronicity of the wound is in part due to the 517 production of elastase by P. aeruginosa, which deteriorates immunoglobulin G and elements of 518 the complement system (Wilson et al., 1998). 519 In diabetes mellitus, the development of foot ulcers is a typical complication of the 520 disease. These patients tend to form nonhealing ulcers on the lower extremities with higher 521 susceptibility than nondiabetic patients (Singh et al., 2005). Insulin treatment has been shown 522 to increase the development of P. aeruginosa biofilms and their antimicrobial tolerance by 523 increasing the levels of eDNA through the lysis of neutrophils and other immune cells in 524 wounds. Furthermore, a direct role of insulin in this prolonged inflammatory response in the 525 wound has been demonstrated in vitro through the use of incremental levels of intracellular c- 526 di-GMP (Watters et al., 2014; Wei et al., 2019). 527 Chronic wound infections are primarily affected by polymicrobial communities, in 528 which P. aeruginosa and S. aureus play a central role (Serra et al., 2015). It has been recently 529 seen that the genotype of the patient influences the microbiome composition of the wound, i.e., 530 depending on the patient’s genetics, certain species are more likely to colonize the wound 531 (Tipton et al., 2020). Genomic analyses have found that genes involved in anaerobic growth, 532 metabolic and energy pathways and membrane integrity are critical for bacterial fitness in 533 wounds (Morgan et al., 2019) 534 535 24 5.3.3. Keratitis 536 Biofilm formation over contact lenses (CLs) is an important cause of corneal infections, 537 as these lenses make direct contact with the corneal surface. The annual incidence rate of 538 microbial keratitis due to CL contamination is approximately 4 per 10000 daily contact lens 539 wearers (Morgan et al., 2005). Biofilms formed over lenses enhance bacterial resistance to 540 antimicrobials (Zegans et al., 2002). P. aeruginosa is a leading cause of corneal infections 541 promoted by CL wearers (Stapleton and Carnt, 2012). Under normal and healthy conditions, the 542 organism can poorly colonize the cornea. Nevertheless, if there is trauma or the cornea is 543 injured, which is often mediated by continuous CL wearing, P. aeruginosa can penetrate the 544 epithelial layer and cause keratitis (Zegans et al., 2002). In the presence of phagocytic cells or 545 corneal epithelial debris, P. aeruginosa can even form denser biofilms on the CL (Burnham et 546 al., 2012; Robertson et al., 2011). While growing in biofilms, bacteria can shift their gene 547 expression to be able to persist in the ocular environment. In this sense, P. aeruginosa adapts to 548 the human corneal epithelium by modulating, mainly, the expression of virulence genes. This 549 corneal-adapted P. aeruginosa forms large biofilm-like aggregates (Evans and Fleiszig, 2013). 550 The T3SS is highly expressed in the adaptation of P. aeruginosa for survival on the corneal 551 surface, and it has an essential role in preserving the biofilm against the attack of host 552 neutrophils. This event leads to NET production, which inhibits the spread of the bacteria to the 553 brain by forming a barrier against the pathogen (Thanabalasuriar et al., 2019). Developing new 554 materials for contact lenses able to inhibit bacterial attachment, viability or the adaptive changes 555 in gene expression associated with bacteria growing on surfaces could have a remarkable impact 556 on reducing the risk of infection (Evans and Fleiszig, 2013). 557 558 5.3.4. Medical device colonization 559 25 P. aeruginosa is a major nosocomial pathogen able to colonize and form perdurable 560 biofilms on indwelling medical devices such as endotracheal tubes (EETs), catheters, and 561 orthopedic implants as well as on the inner surfaces of metal pipes in hospital water systems. P. 562 aeruginosa is responsible for 10-15% of nosocomial infections worldwide (Shi et al., 2019). 563 Biofilm formation has been found in 95% of patients intubated with EET and mechanical 564 ventilation for more than 24 h. ETT biofilms are perdurable and able to remain despite antibiotic 565 treatment. This fact increases the risk of upper respiratory tract infections that eventually lead 566 to ventilator-associated pneumonia (VAP), for which P. aeruginosa is the primary causative 567 agent (Gil-Perotin et al., 2012). Furthermore, the rhamnolipids produced by P. aeruginosa 568 isolates are associated with VAP development (Kohler et al., 2010). P. aeruginosa VAP has 569 increased morbidity and involves intensive care unit stays with the additional cost that they 570 represent (Safdar et al., 2005). 571 Urinary catheters are also susceptible to colonization by P. aeruginosa and lead to 572 catheter-associated urinary tract infections (CAUTIs), the most common hospital-associated 573 infection. P. aeruginosa is responsible for 12% of the CAUTIs acquired in hospitals, which 574 generally tend to be more complicated and have a worse prognosis than those caused by other 575 bacteria (Cole et al., 2014). Iron deficiency in urinary tract infections increases the expression 576 of rhamnolipids in Pseudomonas, promoting biofilm development (Glick et al., 2010). In 577 addition to the increased antimicrobial tolerance that bacteria have when growing in biofilms, 578 many clinical P. aeruginosa isolates from CAUTIs have been shown to encode for multiple 579 antimicrobial resistances (Vipin et al., 2019), complicating the clearance of this infection. 580 581 6. The challenge to reproduce P. aeruginosa polymicrobial chronic infections 582 Even though we now know that biofilms are the predominant type of bacterial growth 583 in nature, planktonic experiments have historically been used as a reference to study chronic 584 32 712 Drug delivery systems (DDS) were created to control the release of therapeutic agents 713 at the target site. As detailed in section 5, P. aeruginosa biofilms’ structure and physiology 714 challenge their treatment and, in this sense, DDS have been of great help. DDS can be used as 715 a surface coating to prevent biofilm formation, as well as carriers to deliver the antimicrobial 716 inside or over the biofilm efficiently. DDS enhance the antimicrobials’ pharmacodynamic and 717 pharmacokinetic effect, increasing the effective concentration in the inner parts of the biofilm 718 for its complete clearance (Liu et al., 2020). Even though different types of DDS have been 719 developed to improve antimicrobial treatment, this review will focus on nanoparticles (NP)- 720 DDS. NPs are an important DDS group, which in continuous development and improvement. 721 Nevertheless, Table 1 also includes other types of DDS (e.g., phage-delivery systems). 722 NPs are DDS widely used for Pseudomonas biofilms’ treatment. NPs are classified 723 depending on the material used in their synthesis. In this sense, they could be: i) metallic NPs, 724 ii) non-metallic NPs, iii) polymeric NPs, iv) lipid-phased NPs, v) ceramic NPs, and vi) quantum 725 dots (Buch et al., 2019). Recently, smart nanomaterials have been developed, which have the 726 particularity that the delivery of the antimicrobial cargo is done in response to a stimulus as, for 727 instance, pH or light (Chen, M. et al., 2019). NPs size is critical to penetrating within the biofilm 728 properly, and it has to range between 5 – 500 nm, being ideally 200 nm the maximum diameter. 729 NPs smaller than 5 nm prone to be filtrated by the kidney while higher than 500 nm to be 730 recognized and cleared by the human complement system (Liu et al., 2019). The shape of the 731 NP is another critical element, as it can influence contact killing as, for instance, it occurs with 732 sharp NPs, which can make a hole in the bacteria and cause cytoplasmic leakage. Otherwise, 733 including mucolytic or other degrading agents in the NPs, together with using electrostatically 734 neutral coating, increase the efficiency of NPs penetrance into the biofilm (Tan et al., 2020). In 735 this line, our group has demonstrated the degrading effect of DNAse coated NPs loaded with 736 33 ciprofloxacin on in vivo-like biofilms of P. aeruginosa, as well as unraveled the effect of specific 737 alginate lyases (Alg2A and A1-II’) as dispersing agents of P. aeruginosa biofilms (Baelo et al., 738 2015; Blanco-Cabra et al., 2020). 739 On the other hand, it has also been detected that cationic nanoparticles can have a good 740 distribution across the biofilm matrix (Baelo et al., 2015; Makabenta et al., 2021). 741 Biocompatibility and cell toxicity are major concerns when using these DDS as depending on 742 the material they are made of, NPs can cause important cytotoxicity, being useless for human 743 treatment. For example, this is one drawback of metallic NPs as high metal doses are toxic for 744 human cells, which is why these NPs are not the preferred choice to use against CF-infections 745 due to the long-term treatment that these infections require (Makabenta et al., 2021; Vandebriel 746 and De Jong, 2012). NPs used in medical applications are usually coated with liposomes, silica 747 or biopolymers to enhance their biocompatibility, as well as to improve their elimination from 748 the body. Poly-D-L-(lactic-co-glycolic acid), polylactic acid, polyethylene glycol, 749 poly(caprolactone, dextran, chitosan, poly(urethanes), poly(ethylene imine) or poly(N- 750 isopropylacrylamide) are some biopolymers frequently used for NPs coating (Tan et al., 2020). 751 An important disadvantage of the current NPs is the amount of cargo that they can load. The 752 vast majority only support low concentrations of the drug, making them suitable as a proof-of- 753 concept but not feasible for clinical application. The high number of NPs needed to achieve 754 efficient antimicrobial concentration limits the balance between antimicrobial activity and 755 cytotoxicity. 756 NPs can load different antibiotics/antimicrobials to be released within the biofilm. 757 Tobramycin, ciprofloxacin, colistin, levofloxacin, amikacin, and gentamicin are known 758 antibiotics delivered by NPs to treat P. aeruginosa infections. Some of them have been used 759 with inhaled systems, although this technology is usually inefficient to eradicate P. aeruginosa 760 biofilm infection (Ho et al., 2019). Otherwise, when using NPs directly against biofilms, it has 761 34 been detected that the small size of these nanocarriers allows the particle diffusion across the 762 thick and dense mucus of CF mucoid biofilms. In this sense, delivery of amikacin antibiotic by 763 nanoscale liposomes has been used to treat P. aeruginosa biofilms in lung infections (Meers et 764 al., 2008). Delivery of nitric oxide (NO) is an additional strategy used against P. aeruginosa 765 biofilms. NO, as endogenous free radical, acts as a broad-spectrum antimicrobial with low 766 toxicity and a great capacity to eradicate biofilms. Silica NPs conjugated with NO have been 767 tested against P. aeruginosa and S. aureus coculture biofilms (Slomberg et al., 2013). Heat 768 generation on NPs has also been seen to improve antibiotics delivery into P. aeruginosa mono 769 and polymicrobial biofilms (Teirlinck et al., 2018). Table 1 summarizes additional strategies 770 employed against P. aeruginosa biofilms using NPs as DDS. 771 772 8. Conclusions 773 P. aeruginosa biofilms are complex structures that become even more intricate when 774 they are formed together with other microorganisms. They represent a clinical and 775 biotechnological burden from different perspectives; therefore, only with the continuous 776 development and improvement of efficient antibiofilm strategies we can tackle the recurrence 777 and chronicity caused by P. aeruginosa biofilm infections. 778 779 Acknowledgments 780 The group is supported by grants from the Ministerio de Economía, Industria y 781 Competitividad, MINECO, and Agencia Estatal de Investigación (AEI), Spain, co-funded by 782 Fondo Europeo de Desarrollo Regional, FEDER, European Union (RTI2018-098573-B-100), 783 the CERCA programme and AGAUR-Generalitat de Catalunya (2017SGR-1079), the European 784 Regional Development Fund (FEDER), Catalan Cystic Fibrosis association and Obra Social 785 “La Caixa”. 786 35 787 Author contributions 788 MC and ET have designed, written and approved the final version of the review. 789 790 36 References 791 Acosta, N., Waddell, B., Heirali, A., Somayaji, R., Surette, M.G., Workentine, M.L., Rabin, 792 H.R., Parkins, M.D., 2020. 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Scheme of the clinically relevant microbial biofilm interactions of P. aeruginosa. 1428 P. aeruginosa can interact and coexist simultaneously with a wide range of microbes from 1429 different phyla. The figure shows the main interactions described for P. aeruginosa with 1430 bacteria (Burkholderia cepacia, Staphylococcus aureus, Prevotella spp., Enterococcus faecalis, 1431 Streptococcus spp., Acinetobacter baumannii, Stenotrophomonas maltophilia, Veillonella spp., 1432 Actinomyces spp., and Propionibacterium spp.), fungi (Aspergillus fumigatus and Candida 1433 albicans) and viruses (respiratory syncytial virus (RSV), human rhinovirus, severe acute 1434 respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza). Many of these interactions 1435 have been found in the disease environment. The figure was created using biorender.com. 1436 1437 Figure 2. Mutual effect of P. aeruginosa and S. aureus pathogenicity and interaction on 1438 each other phenotype. The scheme summarizes the effect that the products of P. aeruginosa 1439 and S. aureus cause on each other, as well as that caused by the products generated as 1440 consequence of their interaction. Blue arrows indicate a beneficial effect, while red lines indicate 1441 an inhibitory effect. In black is specified the mutual effect that the organisms receive as a 1442 consequence of their interaction. Next to each arrow is denoted the action caused by the products 1443 generated, responsible for the inhibitory or beneficial effect promoted to the receptor. 1444 1445 Figure 3. Role of QS on the development of the ECM. The plot shows the effect of the main 1446 circuits of P. aeruginosa QS on the development of the biofilm’s ECM. The principal factors 1447 described affecting QS modulation, with the respective increased autoinducers and the 1448 consequent effect on rhamnolipids (orange), Pel (red), and eDNA (blue) production, are 1449 included in the figure. 1450 1451 50 Figure 4. Main features of P. aeruginosa biofilms and the respective consequences on the 1452 infection progression. The figure shows the major infections caused by P. aeruginosa biofilms: 1453 cystic fibrosis, wounds, infections due to biofilm growth over medical devices, and keratitis 1454 with the representative features of each one. The scheme shows the principal causes that lead to 1455 P. aeruginosa growth in biofilms. Hence, a lung with the alveoli completely filled with an 1456 excess of mucus is presented for CF infections, heart valves, urinary catheters, stents, and 1457 orthopedic devices are shown as examples for medical devices-related infections, a skin wound, 1458 and a foot ulcer are shown for wound infection and, finally, an eye is shown for keratitis 1459 infection. The figure summarizes the main characteristics of each infection detailed through 1460 section 5.3. Created with biorender.com. 1461 Figure 1 Click here to access/download;Figure;New Figure 1.tiff Figure 2 Click here to access/download;Figure;New Figure 2.tiff Figure 3 Click here to access/download;Figure;New Figure 3.tiff Figure 4 Click here to access/download;Figure;New Figure 4.tiff