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Overcoming the challenge of establishing biofilms in vivo: a roadmap for Enterococci
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ScienceDirect Overcoming the challenge of establishing biofilms in vivo: a roadmap for Enterococci Casandra Ai Zhu Tan, Haris Antypas and Kimberly A Kline Enterococcus faecalis forms single and mixed-species biofilms on both tissue and medical devices in the host, often under exposure to fluid flow, giving rise to infections that are recalcitrant to treatment. The factors that drive enterococcal biofilm formation in the host, however, remain unclear. Recent reports in other pathogens show how surface sensing by bacteria can trigger the transition from planktonic to sessile lifestyle. Fluid flow can enhance initial adhesion, but also influence quorum sensing. Biofilm-specific factors, as well as biofilm size and extracellular polymeric substances, can compromise opsonization and phagocytosis. Bacterial interspecies synergy can create favorable conditions in the host for biofilm formation. Through these concepts, we define the knowledge gaps in understanding host-associated E. faecalis biofilm formation and propose a roadmap for future investigations. Address Singapore Centre for Environmental Life Sciences Engineering, School of Biological Sciences, Nanyang Technological University, Singapore Corresponding author: Kline, Kimberly A ([email protected])
Current Opinion in Microbiology 2020, 53:9–18 This review comes from a themed issue on Host-microbe interactions: bacterial Edited by Eric Skaar and Joe P Zackular
https://doi.org/10.1016/j.mib.2020.01.013 1369-5274/ã 2020 Elsevier Ltd. All rights reserved.
Introduction The transition between planktonic and biofilm lifestyles is dictated by environmental cues [1–4]. Understanding how environmental cues in the host drive biofilm formation is essential for improved management of biofilm-related infections. Pathogens such as Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus have recently provided significant insight into how interactions with tissue, fluid flow, immune cells, and other bacterial species in the host can influence bacterial physiology and drive in vivo biofilm formation. In this review, we use these recent findings as a framework to describe key factors involved in biofilmrelated infections caused by the less well-understood Grampositive pathogen Enterococcus faecalis. E. faecalis is the third www.sciencedirect.com
most common pathogen associated with hospital-acquired infections [5]. It accounts for 15% of catheter-associated urinary tract infections (CAUTI) and 5–15% of infective endocarditis (IE) cases, and it is frequently isolated from polymicrobially infected chronic wounds [6–8]. Despite the strong association of biofilm with these serious infections, there is a significant knowledge gap related to in vivo biofilm formation strategies of E. faecalis. In this review, we use recent paradigms from other pathogens to pose hypotheses for how host–microbe interactions drive biofilm formation for E. faecalis.
Fluid flow and surface sensing as mechanical cues for biofilm formation in the host Fluid flow in the host acts as a first barrier for bacterial adhesion [9]. E. faecalis, however, can attach and form biofilm on surfaces exposed to flow of blood or urine, such as the endocardium, urinary tract, and catheters [10]. Adhesion under shear stress is generally attributed to pili, surface proteins, and other surface-exposed polymers [11,12,13,14–17], which are also part of the virulence factor repertoire of E. faecalis [10]. A welldescribed pilus in E. faecalis is the highly conserved, sortase-assembled, endocarditis and biofilm-associated pilus (Ebp) [18]. Ebp pili mediate adhesion to collagen, fibrinogen, and platelets present on a damaged endocardium, leading to biofilm formation and infective endocarditis [19]. In the urinary tract, Ebp pili enhance colonization of the bladder, kidneys, and catheters [20,21]. Ebp-mediated adhesion to catheters is facilitated by fibrinogen deposition, which occurs as a result of inflammation during bladder catheterization [22]. The surface proteins aggregation substance (Agg) and adhesin to collagen from E. faecalis (Ace) also mediate adhesion to extracellular matrix components exposed in damaged endocardium during infective endocarditis [23,24]. Moreover, Ace contributes to both endocarditis and urinary tract infection (UTI) in vivo [25,26]. Similarly, the enterococcal surface protein (Esp) enhances bladder colonization in UTI [27]. Finally, the surfaceassociated polymer lipoteichoic acid (LTA) of E. faecalis contributes to epithelial adhesion [28]. While all of these factors mediate adhesion to host tissues under fluid flow, the effect of shear stress on E. faecalis adhesion has not been reported. Other pathogens exhibit shear stressenhanced adhesion [16,17,29–32]. For instance, shear forces drive the adhesion of S. aureus in infective endocarditis. Staphylococcal cell-surface protein clumping factor A (ClfA) interacts with platelets and forms a thrombus only under high arterial shear conditions Current Opinion in Microbiology 2020, 53:9–18
10 Host-microbe interactions: bacterial
[33]. Moreover, ClfA binds to fibrinogen by a forceactivated mechanism, which enables S. aureus to mediate stronger binding under high physiological shear stress [13]. It is therefore tempting to speculate that enterococcal adhesion factors, such as Ebp pili or Ace, might also mediate shear stress-dependent adhesion. In addition to establishing a foothold in the host, surface adhesion can also serve as a mechanical cue for bacteria that triggers metabolic changes, virulence factor upregulation,
and quorum sensing system expression (Figure 1a,b) [34]. The conversion of this mechanical cue into biochemical changes, known as mechanotransduction, can determine whether attached bacteria will grow into biofilm or return to the planktonic state [35,36]. The surface sensing systems of mechanotransduction are often bacterial appendages, twocomponent systems [37–46], and the cell wall [47] (Table 1). Uropathogenic E. coli (UPEC) senses a surface by binding to mannose residues via the tip adhesin FimH of Type 1 pili, which triggers a feedback loop that further
Figure 1
(a)
(f) Flow
Flow
EPS
Dispersal
Planktonic
(e) AI
Flow
(washed away)
(b) Flow
Surface sensing
AI QS on
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Stages of biofilm development during biofilm-related infections. (a) Biofilm formation begins with planktonic bacteria attaching to host tissue. (b) Initial adhesion may take place under body fluid flow or no-flow conditions, depending on the tissue and site of adherence. During initial adhesion, bacteria sense the surface, which alters their physiology and can prepare them for a transition to a sessile lifestyle. (c) Cell division can generate daughter cells with uneven distribution of intracellular factors, which can result in cell commitment to a biofilm lifestyle or reversal to the planktonic state. (d) As bacteria multiply and form microcolonies, they also start secreting autoinducers (AI). However, AI are washed away at this stage due to fluid flow in the host and consequently, quorum sensing (QS) is not switched on. (e) Once biofilms start maturing, the production of extracellular polymeric substances (EPS) will retain AI within the biofilm, which will activate QS. (f) Eventually, bacteria in mature biofilms can disperse as planktonic bacteria and colonize other locations. Current Opinion in Microbiology 2020, 53:9–18
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Table 1 Examples of bacterial surface sensing factors in mechanotransduction Bacterial Species
Surface sensor
Reference
E. coli
Type 1 pili/FimH adhesin P pili Outer membrane lipoprotein NlpE & Cpx two-component signal transduction pathway Flagellar basal body protein FliL Flagellar motor MotAB Wsp surface sensing system Type 4 pili Cell wall
[37,44,46] [39] [41]
P. mirabilis P. aeruginosa
S. aureus
promotes the expression of Type 1 pili [46]. FimH binding to mannose-coated beads upregulates transcription of fimB. Recombinase FimB promotes the Phase ON orientation of the fimS invertible element, which encodes the fim operon promoter, and enables the expression of Type I pili to further enhance adhesion [48]. Surface sensing in P. aeruginosa is mediated by Type 4 pili (T4P), the flagellar motor MotAB and the Wsp surface sensing system, triggering an intracellular cyclic diguanylate monophosphate (c-di-GMP) increase in a subpopulation of initially attached cells [49,50,51]. This heterogeneous increase results in a high c-di-GMP population that will commit to a sessile lifestyle and a low c-di-GMP population that will commit to surface motility and dispersion, enabling P. aeruginosa to efficiently colonize the host and spread the infection. The heterogeneous c-di-GMP increase might be the result of Wsp system localization on the lateral side of bacteria and the ability of P. aeruginosa to attach either laterally or polarly. Thus, the Wsp system is activated only on laterally attached cells [51], where it mediates phosphorylation of the WspR di-guanylate cyclase, thereby promoting c-di-GMP synthesis [42]. Increased c-di-GMP levels promote adhesion by binding to the cytoplasmic FimW protein, which then rapidly localizes at the cell poles to promote T4P formation [49]. Asymmetric division of high c-di-GMP cells can generate further heterogeneity by producing daughter cells with an uneven distribution of Pch phosphodiesterase (Figure 1c) [49]. This results in a low c-di-GMP flagellated cell that will resume the planktonic lifestyle and a high c-di-GMP piliated cell that will remain attached. In addition to c-di-GMP, surface sensing also regulates cyclic adenosine monophosphate (cAMP) in P. aeruginosa. Mechanical changes in T4P during adhesion are detected by the Pil-Chp chemosensory system, regulating cAMP production and activating cAMP-dependent processes that control adhesion, biofilm formation, and pathogenicity [45]. cAMP also helps P. aeruginosa ‘remember’ surface adhesion, increasing its readiness to re-attach in a subsequent surface encounter [52]. In surface-sentient cells, adhesion memory is imprinted as coupled out-of-phase oscillations of intracellular cAMP levels and T4P activity, which are transferrable to daughter cells. www.sciencedirect.com
[40] [49] [42,51] [45,50,52,62] [47]
By contrast, cAMP levels and T4P activity are not correlated in surface-naı¨ve cells. Overall, these examples show how initial adhesion and surface sensing can trigger intracellular changes to promote a sessile lifestyle. It remains to be determined whether E. faecalis also possesses surface sensing mechanisms to determine the fate of attached bacteria.
The effect of fluid flow, adhesion, and tissue topography on quorum sensing Bacteria in biofilms coordinate group behavior using quorum sensing (QS), an interbacterial communication system based on the production, sensing, and response to extracellular signaling molecules called autoinducers (AI) [53]. In E. faecalis, three QS systems have been identified: the cytolysin, LuxS and Fsr systems [54]. The cytolysin QS system regulates the expression of the pore-forming toxin cytolysin, whereas a role in biofilm formation has been shown for the Fsr and LuxS systems [54]. The significance of the LuxS system in E. faecalis, however, is not yet well-studied. In the case of the Fsr regulatory system, the FsrD propeptide is processed by FsrB to generate the gelatinase biosynthesis-activating pheromone (GBAP), a small lactone that acts as the AI [54]. The membrane sensor kinase FsrC senses the accumulation of GBAP and phosphorylates the response regulator FsrA, which induces expression of gelatinase (GelE) and serine protease (SprE) [54,55,56]. These proteases control autolysin (Atn)-mediated fratricide and extracellular DNA (eDNA) release, which contributes to biofilm formation in vitro [57]. Using a catheter-associated urinary tract infection (CAUTI) murine model, however, E. faecalis established infection and formed biofilm on catheters even in the absence of atn and gelE [58]. GelE is also involved in fibrin degradation and cleavage of Ace, which mediates adhesion to collagen [59,60]. In line with these roles, infection by an E. faecalis gelE mutant in a rabbit model of endocarditis resulted in increased deposition of a fibrinous matrix layer, which suggested that GelE might be involved in bacterial dissemination by Current Opinion in Microbiology 2020, 53:9–18
12 Host-microbe interactions: bacterial
promoting degradation of the vegetation substrate [61]. Despite current knowledge of Fsr-mediated GelE upregulation during biofilm formation and its downstream roles, little is known about the stimuli that induce Fsr QS in the host. Recent studies in P. aeruginosa and S. aureus have shown how surface adhesion and fluid flow can affect QS. Surfaceassociated P. aeruginosa is more sensitive to QS compared to planktonic cells [62]. Surface association sensed by T4P retraction motors and minor pilins induces the sRNA Lrs1, which in turn induces the QS master regulator LasR. Higher levels of LasR in attached cells in the presence of autoinducer N-(3-oxododecanoyl)-homoserine lactone result in increased transcription of downstream QS target genes compared to planktonic P. aeruginosa [62]. The Agr QS system in S. aureus has an inhibitory effect on biofilm formation, favoring biofilm dispersion instead [63]. A recent study found that exposure of S. aureus to fluid flow can reverse this effect by washing away the endogenously produced AIs [64]. A S. aureus strain harboring an AIresponsive agr P3-gfpmut2 transcriptional fusion exhibited fluorescence in the absence but not in the presence of fluid flow during an 8-h exposure. Addition of exogenous AI under flow conditions restored fluorescence, showing that the endogenously produced AIs were washed away by the flow (Figure 1d). During a 16-h exposure to flow, the fluorescence reporter remained inactive in S. aureus cells at the biofilm-fluid interface and as a result biofilm grew thicker. At the same time, cells at the biofilm base started becoming fluorescent, suggesting that the biofilm mass increase protected AI in the inner layers from being washed away (Figure 1e). This population distribution may be beneficial, as Agr-off bacteria exposed to flow can form thicker and more robust biofilms, whereas an Agr-on population in the biofilm base can potentially contribute to dispersal and dissemination (Figure 1f). The same study also showed how complex topography, such as crevices, creates a microenvironment protected from fluid flow to promote AI accumulation and QS activation in S. aureus. A similar effect of fluid flow on QS activity was shown in P. aeruginosa, where increasing the flow rate resulted in a delayed onset of QS signaling [65]. Interestingly, Psl polysaccharide production, which is QS-independent, precedes the onset of QS, suggesting that early extracellular polymeric substance (EPS) production might prevent autoinducer molecules from being washed away. Overall, these findings show how physiology and tissue geometry in the host, as well as the biofilm matrix, are important parameters for QS activity in biofilms. Since E. faecalis also establishes biofilms in the dynamic environments of the heart and the catheterized urinary bladder, we speculate that the degree of exposure to blood and urine flow in combination with surface association and matrix composition may result in a heterogeneous pattern of Fsr activity in the biofilm, similar to what was described for S. aureus and P. aeruginosa. Current Opinion in Microbiology 2020, 53:9–18
Biofilm strategies to overcome innate host immune responses Biofilm-related infections are often chronic, in part due to the inability of the host to clear biofilm [66]. The expression of biofilm-specific immunomodulatory factors, the presence of EPS, and the large size of biofilms together pose a challenge for successful clearance by immune cells. In E. faecalis, among the factors that compromise immune clearance [67], one is also associated with biofilm-formation. GelE, which is regulated by the Fsr QS, facilitates complement resistance by cleaving complement component C5a in vivo in an infective endocarditis model, thus decreasing neutrophil recruitment [61]. Additionally, GelE cleaves complement component C3, which decreases opsonization and phagocytosis in vitro [68]. Independent of its effects on the
Figure 2
(a)
Degrading enzymes ROS Proteases Phagocytosis
Bacteria Neutrophils
Degranulation
Phagolysozyme
50 μm (b)
NETosis Phagocytosis inhibited EPS 50 μm Current Opinion in Microbiology
Evasion of host immune responses in biofilms. (a) As a response to bacterial infection, neutrophils can employ multiple antimicrobial strategies including phagocytosis and subsequent killing in the phagolysosome via degradative enzymes, as well as degranulation, resulting in the release of reactive oxygen species (ROS) and proteases which are microbicidal. Bacteria that escape these antimicrobial strategies can eventually attach to biotic surfaces and initiate biofilm formation. (b) Biofilm aggregates can be resistant to phagocytosis due to their large physical size and extracellular polymeric substances (EPS). Unable to phagocytose biofilms, neutrophils release neutrophil extracellular traps (NETs). However, the role of NETosis remains ambiguous for biofilms, as it selectively helps confine biofilms of some microorganism to the site of infection, yet contributes to biofilm formation of others [87]. www.sciencedirect.com
Biofilm pathogenesis in vivo Tan, Antypas and Kline 13
immune response, GelE also contributes to biofilm formation by regulating Atn-mediated eDNA release [69,70]. These findings together suggest that GelE may play a bifunctional role in biofilm-associated infection, compromising immune clearance while simultaneously enabling E. faecalis biofilm formation. Biofilm-associated factors that compromise complement components have also been described for P. aeruginosa. In P. aeruginosa, LasB elastase can degrade both C1q and C3 complement components [71], but it is also a critical biofilm factor. Deletion of lasB [72] or treatment with LasB inhibitor, N-mercaptoacetyl-Phe-Tyr-amide [73], leads to reduced biofilm formation in vitro in the absence of host cells. Additionally, LasB elastase stimulates exopolysaccharide alginate biosynthesis, which contributes to both structure and antibiotic resistance in P. aeruginosa biofilms [74].
Phagocytosis is critical for innate immune clearance of pathogens. However, biofilm aggregates are usually much larger in size (4 200 mm) [75], compared to planktonic bacteria (1 3 mm), rendering biofilm aggregates resistant to phagocytosis (Figure 2) [76–81]. Moreover, biofilms are encased in a matrix of EPS that may alter the viscoelastic properties of biofilm in a way that it is refractory to phagocytosis [82]. Enterococcal CAUTI and infective endocarditis are good examples of biofilm-related infections that elicit a strong neutrophil response, yet neutrophils alone cannot clear bacteria without concomitant antibiotic treatment [83,84]. Neutrophils in E. faecalis vegetations in a rat endocarditis model release neutrophil extracellular traps (NETs) [83]. However, their effect of NETs on the outcome of the infection, as well as what triggers their release, has not been reported (Figure 2). In
Figure 3
(a)
(b)
Low iron conditions L-ornithine
E. faecalis
Siderophores
E. coli E. coli E. faecalis
Macrophages
(d)
(c)
Phanto-Valentine leucocidin α-hemolysin Methicillin-resistant S. aureus
Siderophores HQNO Rhamnolipids Alginate P. aeruginosa
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Polymicrobial interactions in biofilm-related infections. Interspecies interactions occur during (a) catheter-associated urinary tract infections (CAUTI), (b and c) wound and (d) lung infections. (a) E. faecalis suppresses the host immune response to promote E. coli in CAUTI. (b) E. faecalis exports a cue to increase siderophore expression in E. coli, which promotes E. coli biofilm formation and virulence during wound infections. (c) P. aeruginosa induces expression of virulence factors in methicillin-resistant S. aureus, which leads to delayed wound re-epithelization and healing. (d) Overproduction of alginate by P. aeruginosa reduces production of virulence factors necessary for killing of S. aureus, which promotes S. aureus colonization in lungs of cystic fibrosis patients. www.sciencedirect.com
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Streptococcus mutans and S. aureus endocarditis vegetations, the release of NETs enhanced biofilm formation and expansion, instead of exerting antimicrobial activity [85,86]. Moreover, P. aeruginosa biofilms formed on the cornea of mice are too large to be phagocytized, and neutrophils are unable to penetrate the biofilm, giving rise to an acellular, protective, ‘dead zone’ composed of NETs between the biofilm and neutrophils [87]. The P. aeruginosa cytotoxin ExoS is required for NET release and formation of the dead zone. The P. aeruginosa biofilm EPS component Psl also prevents neutrophil infiltration and promotes the dead zone. Blocking production of both ExoS and Psl results in a switch from neutrophil NET formation to phagocytosis and bacterial killing [87]. A size-dependent NETosis has also been shown for Candida albicans and Mycobacterium bovis, where hyphae and bacterial aggregates but not small yeast or single bacteria triggered NETs release [88]. Although the size and EPS composition of E. faecalis biofilms in vivo have not been sufficiently characterized, it is tempting to speculate that they could play an important role in blocking neutrophils from clearing an enterococcal infection.
Microbe–microbe interactions during infections Interspecies microbe–microbe interactions during biofilmrelated infections can synergistically enhance pathogenesis. E. coli, co-isolated with E. faecalis during CAUTI [89] and wound infection [90,91], derives a benefit and undergoes augmented biofilm formation in the presence of E. faecalis in both CAUTI and wound infection model systems [92,93]. E. faecalis suppresses NF-kB-driven responses in macrophages locally, enabling commensal E. coli to colonize and thus promoting polymicrobial CAUTI (Figure 3a) [93]. Through a different mechanism, export of L-ornithine by E. faecalis signals biosynthesis of the siderophore enterobactin in E. coli to promote E. coli biofilm growth under ironlimiting conditions and in polymicrobial wound infections (Figure 3b) [92]. Enterobactin is crucial in promoting biofilm development, maturation, and survival in E. coli [94], demonstrating how one species can promote the expression of a factor in another species that enables biofilm formation and consequently increases infection severity. In another example, mixed but not single species biofilms of methicillin resistant S. aureus and P. aeruginosa significantly downregulated keratinocyte growth factor 1 (KGF1), resulting in delayed re-epithelization in a porcine wound healing model [95]. In the same model, P. aeruginosa also induced the expression of staphylococcal virulence factors PantonValentine eucocidin and a-hemolysin, increasing the severity of infection (Figure 3c) [95]. S. aureus and P. aeruginosa are also often co-isolated from the lungs of cystic fibrosis (CF) patients. Although P. aeruginosa isolated from monoinfected CF patients outcompetes S. aureus in vitro, P. aeruginosa isolates from co-infected CF patients are more permissive to S. aureus growth [96]. The ability of S. aureus to grow in Current Opinion in Microbiology 2020, 53:9–18
co-infections is due to the overproduction of alginate in P. aeruginosa strains derived from CF co-infections, which in turn reduces the production of factors necessary for robust killing of S. aureus, enabling S. aureus to establish biofilmassociated infections (Figure 3d) [96]. As E. faecalis can often be co-isolated with S. aureus or P. aeruginosa during wound infections [6,97], examining these interspecies interactions will also be important for understanding how these polymicrobial infections initiate and persist.
Conclusions In this review, we selected recent concepts in biofilm pathogenesis from different pathogens and presented them through the lens of E. faecalis biofilm-related infections to define outstanding questions in enterococcal biofilm infection biology. These concepts highlight how mechanical cues from the host and cooperation with other bacterial species alter bacterial physiology and generate a phenotypically heterogeneous population of biofilm-associated and planktonic bacteria to guarantee survival against the immune system and infection dissemination. Lessons learned from these studies demonstrate the importance of investigating how E. faecalis responds to fluid flow and surface adhesion and how these cues might trigger physiological changes to direct the transition between planktonic and biofilm state. Exploring the EPS composition, as well as the biofilm interplay with immune cells, will also help us to understand why E. faecalis biofilms are not cleared by the immune system. Given that E. faecalis is often co-isolated with other pathogens, it will be important to mechanistically understand whether and how interspecies interactions affect pathogenesis. Characterization of host– biofilm interactions with spatiotemporal and single-cell resolution using new in vitro microfluidic models, together with animal models and omics technologies, will help us open the black box of enterococcal biofilm pathogenesis and ultimately design new diagnostic and treatment strategies.
Conflict of interest Nothing to declare.
CRediT authorship contribution statement Casandra Ai Zhu Tan: Conceptualization, Writing original draft, Writing - review & editing, Visualization. Haris Antypas: Conceptualization, Writing - original draft, Writing - review & editing. Kimberly A Kline: Conceptualization, Writing - review & editing, Supervision, Funding acquisition.
Acknowledgements Work in the Kline laboratory related to this article is supported by the Ministry of Education Singapore under its Research Centre of Excellence Program and by the Ministry of Education Singapore under its Tier 2 programme (MOE2018-T2-1-127) and the National Medical Research Council (OFIRG18may-0039). Preparation of this review article was also financially supported by Knut and Alice Wallenberg Foundation under its Wallenberg-NTU postdoctoral fellowship programme (to H.A.) and the Interdisciplinary Graduate Programme of Nanyang Technological University (to C.A.Z.T.). Figures in this article are created with BioRender. com. www.sciencedirect.com
Biofilm pathogenesis in vivo Tan, Antypas and Kline 15
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Gambino M, Cappitelli F: Mini-review: biofilm responses to oxidative stress. Biofouling 2016, 32:167-178.
2.
Kavanaugh JS, Horswill AR: Impact of environmental cues on Staphylococcal quorum sensing and biofilm development. J Biol Chem 2016, 291:12556-12564.
3.
Tolker-Nielsen T: Biofilm development. Microbiol Spectr 2015, 3 MB-0001-2014.
4.
Toyofuku M, Inaba T, Kiyokawa T, Obana N, Yawata Y, Nomura N: Environmental factors that shape biofilm formation. Biosci Biotechnol Biochem 2016, 80:7-12.
5.
Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK: Antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006–2007. Infect Control Hosp Epidemiol 2008, 29:996-1011.
6.
Gjødsbøl K, Christensen JJ, Karlsmark T, Jørgensen B, Klein BM, Krogfelt KA: Multiple bacterial species reside in chronic wounds: a longitudinal study. Int Wound J 2006, 3:225-231.
7.
Peng D, Li X, Liu P, Luo M, Chen S, Su K, Zhang Z, He Q, Qiu J, Li Y: Epidemiology of pathogens and antimicrobial resistance of catheter-associated urinary tract infections in intensive care units: a systematic review and meta-analysis. Am J Infect Control 2018, 46:e81-e90.
8.
Agudelo Higuita NI, Huycke MM: Enterococcal disease, epidemiology, and implications for treatment. In Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Edited by Gilmore MS, Clewell DB, Ike Y, Shankar N. Massachusetts Eye and Ear Infirmary; 2014.
9.
Tonkin ML, Boulanger MJ: The shear stress of host cell invasion: exploring the role of biomolecular complexes. PLoS Pathog 2015, 11:e1004539.
10. Ch’ng J-H, Chong KK, Lam LN, Wong JJ, Kline KA: Biofilm associated infection by enterococci. Nat Rev Microbiol 2019, 17:82-94 A comprehensive review of E. faecalis and E. faecium pathogenesis with a focus on biofilm-related infections, persistence, polymicrobial biofilms and novel treatment strategies. 11. Beaussart A, Baker AE, Kuchma SL, El-Kirat-Chatel S, O’Toole GA, Dufreˆne YF: Nanoscale adhesion forces of Pseudomonas aeruginosa type IV pili. ACS Nano 2014, 8:1072310733. 12. Claes J, Liesenborghs L, Peetermans M, Veloso T, Missiakas D, Schneewind O, Mancini S, Entenza J, Hoylaerts M, Heying R: Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall. J Thromb Haemost 2017, 15:1009-1019. 13. Herman-Bausier P, Labate C, Towell AM, Derclaye S, Geoghegan JA, Dufreˆne YF: Staphylococcus aureus clumping factor A is a force-sensitive molecular switch that activates bacterial adhesion. Proc Natl Acad Sci U S A 2018, 115:55645569 The authors elucidated the mechanistic details underlying staphylococcal ClfA adhesion to fibrinogen, showing a force-activated mechanism similar to the E. coli catch bond, which explains how S. aureus adheres and causes an infection to the host under high shear stress. 14. Mairey E, Genovesio A, Donnadieu E, Bernard C, Jaubert F, Pinard E, Seylaz J, Olivo-Marin J-C, Nassif X, Dume´nil G: Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood–brain barrier. J Exp Med 2006, 203:1939-1950. 15. Melican K, Sandoval RM, Kader A, Josefsson L, Tanner GA, Molitoris BA, Richter-Dahlfors A: Uropathogenic Escherichia coli P and Type 1 fimbriae act in synergy in a living host to facilitate www.sciencedirect.com
renal colonization leading to nephron obstruction. PLoS Pathog 2011, 7:e1001298. 16. Pappelbaum KI, Gorzelanny C, Gra¨ssle S, Suckau J, Laschke MW, Bischoff M, Bauer C, Schorpp-Kistner M, Weidenmaier C, Schneppenheim R: Ultralarge von Willebrand factor fibers mediate luminal Staphylococcus aureus adhesion to an intact endothelial cell layer under shear stress. Circulation 2013, 128:50-59. 17. Thomas WE, Nilsson LM, Forero M, Sokurenko EV, Vogel V: Shear-dependent ‘stick-and-roll’ adhesion of type 1 fimbriated Escherichia coli. Mol Microbiol 2004, 53:1545-1557. 18. Nallapareddy SR, Sillanpa¨a¨ J, Mitchell J, Singh KV, Chowdhury SA, Weinstock GM, Sullam PM, Murray BE: Conservation of Ebp-type pilus genes among Enterococci and demonstration of their role in adherence of Enterococcus faecalis to human platelets. Infect Immun 2011, 79:2911-2920. 19. Nallapareddy SR, Singh KV, Sillanpa¨a¨ J, Zhao M, Murray BE: Relative contributions of Ebp pili and the collagen adhesin Ace to host extracellular matrix protein adherence and experimental urinary tract infection by Enterococcus faecalis OG1RF. Infect Immun 2011, 79:2901-2910. 20. Nielsen HV, Guiton PS, Kline KA, Port GC, Pinkner JS, Neiers F, Normark S, Henriques-Normark B, Caparon MG, Hultgren SJ: The metal ion-dependent adhesion site motif of the Enterococcus faecalis EbpA pilin mediates pilus function in catheterassociated urinary tract infection. mBio 2012, 3 e00177-12. 21. Singh KV, Nallapareddy SR, Murray BE: Importance of the ebp (endocarditisand biofilm-associated pilus) locus in the pathogenesis of Enterococcus faecalis ascending urinary tract infection. J Infect Dis 2007, 195:1671-1677. 22. Flores-Mireles AL, Walker JN, Bauman TM, Potretzke AM, Schreiber HL, Park AM, Pinkner JS, Caparon MG, Hultgren SJ, Desai A: Fibrinogen release and deposition on urinary catheters placed during urological procedures. J Urol 2016, 196:416-421. 23. Nallapareddy SR, Qin X, Weinstock GM, Ho¨o¨k M, Murray BE: Enterococcus faecalis adhesin, ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I. Infect Immun 2000, 68:5218-5224. 24. Rozdzinski E, Marre R, Susa M, Wirth R, Muscholl-Silberhorn A: Aggregation substance-mediated adherence of Enterococcus faecalis to immobilized extracellular matrix proteins. Microb Pathog 2001, 30:211-220. 25. Lebreton F, Riboulet-Bisson E, Serror P, Sanguinetti M, Posteraro B, Torelli R, Hartke A, Auffray Y, Giard J-C: ace, which encodes an adhesin in Enterococcus faecalis, is regulated by Ers and is involved in virulence. Infect Immun 2009, 77:28322839. 26. Singh KV, Nallapareddy SR, Sillanpa¨a¨ J, Murray BE: Importance of the collagen adhesin Ace in pathogenesis and protection against Enterococcus faecalis experimental endocarditis. PLoS Pathog 2010, 6:e1000716. 27. Shankar N, Lockatell CV, Baghdayan AS, Drachenberg C, Gilmore MS, Johnson DE: Role of Enterococcus faecalis surface protein ESP in the pathogenesis of ascending urinary tract infection. Infect Immun 2001, 69:4366-4372. 28. Fabretti F, Theilacker C, Baldassarri L, Kaczynski Z, Kropec A, Holst O, Huebner J: Alanine esters of Enterococcal lipoteichoic acid play a role in biofilm formation and resistance to antimicrobial peptides. Infect Immun 2006, 74:4164-4171. 29. Forero M, Yakovenko O, Sokurenko EV, Thomas WE, Vogel V: Uncoiling mechanics of Escherichia coli type I fimbriae are optimized for catch bonds. PLoS Biol 2006, 4:e298. 30. Lecuyer S, Rusconi R, Shen Y, Forsyth A, Vlamakis H, Kolter R, Stone HA: Shear stress increases the residence time of adhesion of Pseudomonas aeruginosa. Biophys J 2011, 100:341-350. 31. Thomas W, Forero M, Yakovenko O, Nilsson L, Vicini P, Sokurenko E, Vogel V: Catch-bond model derived from allostery Current Opinion in Microbiology 2020, 53:9–18
16 Host-microbe interactions: bacterial
explains force-activated bacterial adhesion. Biophys J 2006, 90:753-764. 32. Weaver WM, Dharmaraja S, Milisavljevic V, Di Carlo D: The effects of shear stress on isolated receptor–ligand interactions of Staphylococcus epidermidis and human plasma fibrinogen using molecularly patterned microfluidics. Lab Chip 2011, 11:883-889. 33. Kerrigan SW, Loughmann A, Meade G, Foster TJ, Cox D: Staphylococcus aureus clumping factor mediates rapid thrombus formation under high shear. Blood 2006, 108:1816. 34. Harapanahalli AK, Younes JA, Allan E, van der Mei HC, Busscher HJ: Chemical signals and mechanosensing in bacterial responses to their environment. PLoS Pathog 2015, 11:e1005057. 35. Persat A: Bacterial mechanotransduction. Curr Opin Microbiol 2017, 36:1-6. 36. Stones DH, Krachler AM: Against the tide: the role of bacterial adhesion in host colonization. Biochem Soc Trans 2016, 44:1571-1580. 37. Otto K, Norbeck J, Larsson T, Karlsson K-A, Hermansson M: Adhesion of type 1-fimbriated Escherichia coli to abiotic surfaces leads to altered composition of outer membrane proteins. J Bacteriol 2001, 183:2445-2453. 38. Lele PP, Hosu BG, Berg HC: Dynamics of mechanosensing in the bacterial flagellar motor. Proc Natl Acad Sci U S A 2013, 110:11839-11844. 39. Zhang JP, Normark S: Induction of gene expression in Escherichia coli after pilus-mediated adherence. Science 1996, 273:1234-1236. 40. Lee Y-Y, Belas R: Loss of FliL alters Proteus mirabilis surface sensing and temperature-dependent swarming. J Bacteriol 2015, 197:159-173. 41. Otto K, Silhavy TJ: Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc Natl Acad Sci U S A 2002, 99:2287-2292. 42. O’Connor JR, Kuwada NJ, Huangyutitham V, Wiggins PA, Harwood CS: Surface sensing and lateral subcellular localization of WspA, the receptor in a chemosensory-like system leading to c-di-GMP production. Mol Microbiol 2012, 86:720-729. 43. Kansal R, Rasko DA, Sahl JW, Munson GP, Roy K, Luo Q, Sheikh A, Kuhne KJ, Fleckenstein JM: Transcriptional modulation of enterotoxigenic Escherichia coli virulence genes in response to epithelial cell interactions. Infect Immun 2013, 81:259-270.
Pseudomonas aeruginosa. Cell Host Microbe 2019, 25:140-152. e6 The authors showed how the flagellar motor mediates surface sensing in P. aeruginosa, triggering polar localization of FimW bound by c-di-GMP and in turn leading to Type 4 pili assembly and enhanced adhesion. Adhesion is followed by an asymmetric division, which generates two daughter cells with high and low levels of c-di-GMP that gave rise to a sessile and a planktonic cell respectively. This single-cell study shows how surface sensing can alter physiology and prepare bacteria for a sessile lifestyle. Moreover, it demonstrates the phenomenon of bacterial labour division, which serves as an efficient strategy for uniform host colonization. 50. Rodesney CA, Roman B, Dhamani N, Cooley BJ, Katira P, Touhami A, Gordon VD: Mechanosensing of shear by Pseudomonas aeruginosa leads to increased levels of the cyclic-di-GMP signal initiating biofilm development. Proc Natl Acad Sci U S A 2017, 114:5906-5911 This article showed how Pel polysaccharide production and shear stress increases friction between attached P. aeruginosa and a surface and leads to an increase in c-di-GMP intracellular levels. This type of surface sensing requires fully functional Type 4 pili. 51. Armbruster CR, Lee CK, Parker-Gilham J, de Anda J, Xia A, Zhao K, Murakami K, Tseng BS, Hoffman LR, Jin F: Heterogeneity in surface sensing suggests a division of labor in Pseudomonas aeruginosa populations. eLife 2019, 8:e45084 The authors studied c-di-GMP production in single surface-associated P. aeruginosa and identified a high c-di-GMP subpopulation committed to biofilm formation and a low c-di-GMP subpopulation committed to surface motility. This heterogeneity was linked to the different activity levels of the Wsp surface sensing system among attached bacteria. 52. Lee CK, de Anda J, Baker AE, Bennett RR, Luo Y, Lee EY, Keefe JA, Helali JS, Ma J, Zhao K: Multigenerational memory and adaptive adhesion in early bacterial biofilm communities. Proc Natl Acad Sci U S A 2018, 115:4471-4476 The authors identified a “learning-by-doing” mechanism in P. aeruginosa, in which bacteria remember surface adhesion events over several generations, increasing their readiness to re-attach compared to surfacenaı¨ve cells. This memory is based on coupled out-of-phase oscillations of cAMP levels and Type 4 pili activity. 53. Rutherford ST, Bassler BL: Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med 2012, 2:a012427. 54. Ali L, Goraya MU, Arafat Y, Ajmal M, Chen J-L, Yu D: Molecular mechanism of quorum-sensing in Enterococcus faecalis: its role in virulence and therapeutic approaches. Int J Mol Sci 2017, 18(5):960 In this review, recent literature about the cytolysin, LuxS, Fsr quorum sensing systems is discussed, as well as their role in enterococcal infections (a comprehensive table is included with relevant references), and potential therapeutic strategies.
44. Bhomkar P, Materi W, Semenchenko V, Wishart DS: Transcriptional response of E. coli upon FimH-mediated fimbrial adhesion. Gene Regul Syst Biol 2010, 4:1-17.
55. Qin X, Singh KV, Weinstock GM, Murray BE: Characterization of fsr, a regulator controlling expression of gelatinase and serine protease in Enterococcus faecalis OG1RF. J Bacteriol 2001, 183:3372-3382.
45. Persat A, Inclan YF, Engel JN, Stone HA, Gitai Z: Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2015, 112:7563-7568.
56. Qin X, Singh KV, Weinstock GM, Murray BE: Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence. Infect Immun 2000, 68:2579-2586.
46. Schwan WR, Beck MT, Hung CS, Hultgren SJ: Differential regulation of Escherichia coli fim genes following binding to mannose receptors. J Pathog 2018, 2018:2897581 This study shows how the FimH-mannose interaction upregulates transcription of recombinase fimB and downregulates transcription of recombinase fimE, which shifted the fimS promoter to phase ON and upregulated expression of Type I pili.
57. Thomas VC, Hiromasa Y, Harms N, Thurlow L, Tomich J, Hancock LE: A fratricidal mechanism is responsible for eDNA release and contributes to biofilm development of Enterococcus faecalis. Mol Microbiol 2009, 72:1022-1036.
47. Harapanahalli AK, Chen Y, Li J, Busscher HJ, van der Mei HC: Influence of adhesion force on icaA and cidA gene expression and production of matrix components in Staphylococcus aureus biofilms. Appl Environ Microbiol 2015, 81:3369-3378.
58. Guiton PS, Hung CS, Hancock LE, Caparon MG, Hultgren SJ: Enterococcal biofilm formation and virulence in an optimized murine model of foreign body-associated urinary tract infections. Infect Immun 2010, 78:4166-4175.
48. Schwan WR: Regulation of fim genes in uropathogenic Escherichia coli. World J Clin Infect Dis 2011, 1:17-25.
59. Pinkston KL, Gao P, Diaz-Garcia D, Sillanpa¨a¨ J, Nallapareddy SR, Murray BE, Harvey BR: The Fsr quorum-sensing system of Enterococcus faecalis modulates surface display of the collagen-binding MSCRAMM Ace through regulation of gelE. J Bacteriol 2011, 193:4317-4325.
49. Laventie B-J, Sangermani M, Estermann F, Manfredi P, Planes R, Hug I, Jaeger T, Meunier E, Broz P, Jenal U: A surface-induced asymmetric program promotes tissue colonization by
60. Waters CM, Antiporta MH, Murray BE, Dunny GM: Role of the Enterococcus faecalis GelE protease in determination of cellular chain length, supernatant pheromone levels, and
Current Opinion in Microbiology 2020, 53:9–18
www.sciencedirect.com
Biofilm pathogenesis in vivo Tan, Antypas and Kline 17
degradation of fibrin and misfolded surface proteins. J Bacteriol 2003, 185:3613-3623.
phagocytosis of Streptococcus pneumoniae. Infect Immun 2013, 81:2606-2615.
61. Thurlow LR, Thomas VC, Narayanan S, Olson S, Fleming SD, Hancock LE: Gelatinase contributes to the pathogenesis of endocarditis caused by Enterococcus faecalis. Infect Immun 2010, 78:4936-4943.
78. Horvath DJ Jr, Li B, Casper T, Partida-Sanchez S, Hunstad DA, Hultgren SJ, Justice SS: Morphological plasticity promotes resistance to phagocyte killing of uropathogenic Escherichia coli. Microbes Infect 2011, 13:426-437.
62. Chuang SK, Vrla GD, Fro¨hlich KS, Gitai Z: Surface association sensitizes Pseudomonas aeruginosa to quorum sensing. Nat Commun 2019, 10:1-10 This study shows how surface association can lead to the upregulation of quorum sensing master regulator LasR via sRNA Lrs1. Consequently, surface-associated cells can achieve higher levels of quorum sensing activity compared to planktonic cells in the presence of autoinducer N-(3oxododecanoyl)-homoserine lactone.
79. Jesaitis AJ, Franklin MJ, Berglund D, Sasaki M, Lord CI, Bleazard JB, Duffy JE, Beyenal H, Lewandowski Z: Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions. J Immunol 2003, 171:4329-4339.
63. Boles BR, Horswill AR: Agr-mediated dispersal of Staphylococcus aureus biofilms. PLoS Pathog 2008, 4: e1000052.
80. Thurlow LR, Hanke ML, Fritz T, Angle A, Aldrich A, Williams SH, Engebretsen IL, Bayles KW, Horswill AR, Kielian T: Staphylococcus aureus biofilms prevent macrophage phagocytosis and attenuate inflammation in vivo. J Immunol 2011, 186:6585-6596.
64. Kim MK, Ingremeau F, Zhao A, Bassler BL, Stone HA: Local and global consequences of flow on bacterial quorum sensing. Nat Microbiol 2016, 1:15005.
81. van Gennip M, Christensen LD, Alhede M, Qvortrup K, Jensen PØ, Høiby N, Givskov M, Bjarnsholt T: Interactions between polymorphonuclear leukocytes and Pseudomonas aeruginosa biofilms on silicone implants in vivo. Infect Immun 2012, 80:2601-2607.
65. Emge P, Moeller J, Jang H, Rusconi R, Yawata Y, Stocker R, Vogel V: Resilience of bacterial quorum sensing against fluid flow. Sci Rep 2016, 6:33115.
82. Stewart PS: Biophysics of biofilm infection. Pathog Dis 2014, 70:212-218.
66. Bjarnsholt T: The role of bacterial biofilms in chronic infections. APMIS Suppl 2013, 121:1-58.
83. Augustin P, Alsalih G, Launey Y, Delbosc S, Louedec L, Ollivier V, Chau F, Montravers P, Duval X, Michel JB: Predominant role of host proteases in myocardial damage associated with infectious endocarditis induced by Enterococcus faecalis in a rat model. Infect Immun 2013, 81:1721-1729.
67. Kao PHN, Kline KA: Jekyll and Mr. Hide: how Enterococcus faecalis subverts the host immune response to cause infection. J Mol Biol 2019, 431:2932-2945 An extensive review on how E. faecalis transits from a commensal bacterium to a pathogen and the strategies it adopts to suppress or evade the host innate and adaptive immune responses elicited during infections.
84. Rousseau M, Goh HS, Holec S, Albert ML, Williams RB, Ingersoll MA, Kline KA: Bladder catheterization increases susceptibility to infection that can be prevented by prophylactic antibiotic treatment. JCI Insight 2016, 1:e88178.
68. Park SY, Shin YP, Kim CH, Park HJ, Seong YS, Kim BS, Seo SJ, Lee IH: Immune evasion of Enterococcus faecalis by an extracellular gelatinase that cleaves C3 and iC3b. J Immunol 2008, 181:6328-6336.
85. Jung CJ, Yeh CY, Hsu RB, Lee CM, Shun CT, Chia JS: Endocarditis pathogen promotes vegetation formation by inducing intravascular neutrophil extracellular traps through activated platelets. Circulation 2015, 131:571-581.
69. Kristich CJ, Li Y-H, Cvitkovitch DG, Dunny GM: Esp-independent biofilm formation by Enterococcus faecalis. J Bacteriol 2004, 186:154-163.
86. Hsu CC, Hsu RB, Ohniwa RL, Chen JW, Yuan CT, Chia JS, Jung CJ: Neutrophil extracellular traps enhance Staphylococcus aureus vegetation formation through interaction with platelets in infective endocarditis. Thromb Haemost 2019, 119:786-796.
70. Thomas VC, Thurlow LR, Boyle D, Hancock LE: Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development. J Bacteriol 2008, 190:5690-5698. 71. Hong Y, Ghebrehiwet B: Effect of Pseudomonas aeruginosa elastase and alkaline protease on serum complement and isolated components C1q and C3. Clin Immunol Immunopathol 1992, 62:133-138. 72. Yu H, He X, Xie W, Xiong J, Sheng H, Guo S, Huang C, Zhang D, Zhang K: Elastase LasB of Pseudomonas aeruginosa promotes biofilm formation partly through rhamnolipidmediated regulation. Can J Microbiol 2014, 60:227-235. 73. Cathcart GR, Quinn D, Greer B, Harriott P, Lynas JF, Gilmore BF, Walker B: Novel inhibitors of the Pseudomonas aeruginosa virulence factor LasB: a potential therapeutic approach for the attenuation of virulence mechanisms in pseudomonal infection. Antimicrob Agents Chemother 2011, 55:2670-2678. 74. Hentzer M, Teitzel GM, Balzer GJ, Heydorn A, Molin S, Givskov M, Parsek MR: Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J Bacteriol 2001, 183:5395-5401. 75. Bjarnsholt T, Alhede M, Alhede M, Eickhardt-Sørensen SR, Moser C, Ku¨hl M, Jensen PØ, Høiby N: The in vivo biofilm. Trends Microbiol 2013, 21:466-474. 76. Bernut A, Herrmann J-L, Kissa K, Dubremetz J-F, Gaillard J-L, Lutfalla G, Kremer L: Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation. Proc Natl Acad Sci U S A 2014, 111:E943-E952. 77. Domenech M, Ramos-Sevillano E, Garcı´a E, Moscoso M, Yuste J: Biofilm formation avoids complement immunity and www.sciencedirect.com
87. Thanabalasuriar A, Scott BNV, Peiseler M, Willson ME, Zeng Z, Warrener P, Keller AE, Surewaard BGJ, Dozier EA, Korhonen JT: Neutrophil extracellular traps confine Pseudomonas aeruginosa ocular biofilms and restrict brain invasion. Cell Host Microbe 2019, 25:526-536.e4 The authors investigated P. aeruginosa pathogenesis in keratitis and found that neutrophils release NETs at the base of the biofilms and is dependent on the T3SS of P. aeruginosa, which in turn prevents dissemination of P. aeruginosa into the brain. Blocking both T3SS PcrV and exopolysaccharide Psl with antibodies enables neutrophils to break down biofilms by transiting from NETosis to intracellular phagocytosis and killing. 88. Branzk N, Lubojemska A, Hardison SE, Wang Q, Gutierrez MG, Brown GD, Papayannopoulos V: Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol 2014, 15:1017-1025. 89. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ: Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 2015, 13:269-284. 90. Citron DM, Goldstein EJ, Merriam CV, Lipsky BA, Abramson MA: Bacteriology of moderate-to-severe diabetic foot infections and in vitro activity of antimicrobial agents. J Clin Microbiol 2007, 45:2819-2828. 91. Giacometti A, Cirioni O, Schimizzi A, Del Prete M, Barchiesi F, D’errico M, Petrelli E, Scalise G: Epidemiology and microbiology of surgical wound infections. J Clin Microbiol 2000, 38:918-922. 92. Keogh D, Tay W, Ho Y, Dale J, Chen S, Umashankar S, Williams RH, Chen S, Dunny G, Kline K: Enterococcal metabolite cues facilitate interspecies niche modulation and polymicrobial infection. Cell Host Microbe 2016, 20:493-503. Current Opinion in Microbiology 2020, 53:9–18
18 Host-microbe interactions: bacterial
93. Tien BYQ, Goh HMS, Chong KKL, Bhaduri-Tagore S, Holec S, Dress R, Ginhoux F, Ingersoll MA, Williams RB, Kline KA: Enterococcus faecalis promotes innate immune suppression and polymicrobial catheter-associated urinary tract infection. Infect Immun 2017, 85:e00378-00317 This paper shows that E. faecalis suppresses macrophage activation via NF-kB, which in turn promotes commensal E. coli strain colonization in a polymicrobial CAUTI model.
Pseudomonas aeruginosa in polymicrobial wound infection. PLoS One 2013, 8:e56846.
94. May T, Okabe S: Enterobactin is required for biofilm development in reduced-genome Escherichia coli. Environ Microbiol 2011, 13:3149-3162.
96. Limoli DH, Whitfield GB, Kitao T, Ivey ML, Davis MR, Grahl N, Hogan DA, Rahme LG, Howell PL, O’Toole GA: Pseudomonas aeruginosa alginate overproduction promotes coexistence with Staphylococcus aureus in a model of cystic fibrosis respiratory infection. mBio 2017, 8 e00186-00117 The authors found that P. aeruginosa with a mucoid phenotype due to overproduction of alginate, reduces the production of virulence factors which are needed for killing of S. aureus and hence enabling the coexistence between the two species in a cystic fibrosis respiratory infection model.
95. Pastar I, Nusbaum AG, Gil J, Patel SB, Chen J, Valdes J, Stojadinovic O, Plano LR, Tomic-Canic M, Davis SC: Interactions of methicillin resistant Staphylococcus aureus USA300 and
97. James GA, Swogger E, Wolcott R, Pulcini Ed, Secor P, Sestrich J, Costerton JW, Stewart PS: Biofilms in chronic wounds. Wound Repair Regen 2008, 16:37-44.
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ScienceDirect Overcoming the challenge of establishing biofilms in vivo: a roadmap for Enterococci Casandra Ai Zhu Tan, Haris Antypas and Kimberly A Kline Enterococcus faecalis forms single and mixed-species biofilms on both tissue and medical devices in the host, often under exposure to fluid flow, giving rise to infections that are recalcitrant to treatment. The factors that drive enterococcal biofilm formation in the host, however, remain unclear. Recent reports in other pathogens show how surface sensing by bacteria can trigger the transition from planktonic to sessile lifestyle. Fluid flow can enhance initial adhesion, but also influence quorum sensing. Biofilm-specific factors, as well as biofilm size and extracellular polymeric substances, can compromise opsonization and phagocytosis. Bacterial interspecies synergy can create favorable conditions in the host for biofilm formation. Through these concepts, we define the knowledge gaps in understanding host-associated E. faecalis biofilm formation and propose a roadmap for future investigations. Address Singapore Centre for Environmental Life Sciences Engineering, School of Biological Sciences, Nanyang Technological University, Singapore Corresponding author: Kline, Kimberly A ([email protected])
Current Opinion in Microbiology 2020, 53:9–18 This review comes from a themed issue on Host-microbe interactions: bacterial Edited by Eric Skaar and Joe P Zackular
https://doi.org/10.1016/j.mib.2020.01.013 1369-5274/ã 2020 Elsevier Ltd. All rights reserved.
Introduction The transition between planktonic and biofilm lifestyles is dictated by environmental cues [1–4]. Understanding how environmental cues in the host drive biofilm formation is essential for improved management of biofilm-related infections. Pathogens such as Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus have recently provided significant insight into how interactions with tissue, fluid flow, immune cells, and other bacterial species in the host can influence bacterial physiology and drive in vivo biofilm formation. In this review, we use these recent findings as a framework to describe key factors involved in biofilmrelated infections caused by the less well-understood Grampositive pathogen Enterococcus faecalis. E. faecalis is the third www.sciencedirect.com
most common pathogen associated with hospital-acquired infections [5]. It accounts for 15% of catheter-associated urinary tract infections (CAUTI) and 5–15% of infective endocarditis (IE) cases, and it is frequently isolated from polymicrobially infected chronic wounds [6–8]. Despite the strong association of biofilm with these serious infections, there is a significant knowledge gap related to in vivo biofilm formation strategies of E. faecalis. In this review, we use recent paradigms from other pathogens to pose hypotheses for how host–microbe interactions drive biofilm formation for E. faecalis.
Fluid flow and surface sensing as mechanical cues for biofilm formation in the host Fluid flow in the host acts as a first barrier for bacterial adhesion [9]. E. faecalis, however, can attach and form biofilm on surfaces exposed to flow of blood or urine, such as the endocardium, urinary tract, and catheters [10]. Adhesion under shear stress is generally attributed to pili, surface proteins, and other surface-exposed polymers [11,12,13,14–17], which are also part of the virulence factor repertoire of E. faecalis [10]. A welldescribed pilus in E. faecalis is the highly conserved, sortase-assembled, endocarditis and biofilm-associated pilus (Ebp) [18]. Ebp pili mediate adhesion to collagen, fibrinogen, and platelets present on a damaged endocardium, leading to biofilm formation and infective endocarditis [19]. In the urinary tract, Ebp pili enhance colonization of the bladder, kidneys, and catheters [20,21]. Ebp-mediated adhesion to catheters is facilitated by fibrinogen deposition, which occurs as a result of inflammation during bladder catheterization [22]. The surface proteins aggregation substance (Agg) and adhesin to collagen from E. faecalis (Ace) also mediate adhesion to extracellular matrix components exposed in damaged endocardium during infective endocarditis [23,24]. Moreover, Ace contributes to both endocarditis and urinary tract infection (UTI) in vivo [25,26]. Similarly, the enterococcal surface protein (Esp) enhances bladder colonization in UTI [27]. Finally, the surfaceassociated polymer lipoteichoic acid (LTA) of E. faecalis contributes to epithelial adhesion [28]. While all of these factors mediate adhesion to host tissues under fluid flow, the effect of shear stress on E. faecalis adhesion has not been reported. Other pathogens exhibit shear stressenhanced adhesion [16,17,29–32]. For instance, shear forces drive the adhesion of S. aureus in infective endocarditis. Staphylococcal cell-surface protein clumping factor A (ClfA) interacts with platelets and forms a thrombus only under high arterial shear conditions Current Opinion in Microbiology 2020, 53:9–18
10 Host-microbe interactions: bacterial
[33]. Moreover, ClfA binds to fibrinogen by a forceactivated mechanism, which enables S. aureus to mediate stronger binding under high physiological shear stress [13]. It is therefore tempting to speculate that enterococcal adhesion factors, such as Ebp pili or Ace, might also mediate shear stress-dependent adhesion. In addition to establishing a foothold in the host, surface adhesion can also serve as a mechanical cue for bacteria that triggers metabolic changes, virulence factor upregulation,
and quorum sensing system expression (Figure 1a,b) [34]. The conversion of this mechanical cue into biochemical changes, known as mechanotransduction, can determine whether attached bacteria will grow into biofilm or return to the planktonic state [35,36]. The surface sensing systems of mechanotransduction are often bacterial appendages, twocomponent systems [37–46], and the cell wall [47] (Table 1). Uropathogenic E. coli (UPEC) senses a surface by binding to mannose residues via the tip adhesin FimH of Type 1 pili, which triggers a feedback loop that further
Figure 1
(a)
(f) Flow
Flow
EPS
Dispersal
Planktonic
(e) AI
Flow
(washed away)
(b) Flow
Surface sensing
AI QS on
EPS
AI (retained)
Inital adhesion
Biofilm maturation (d) Flow
(c)
AI Flow
(washed away)
AI QS off
Cell division
Microcolony formation Current Opinion in Microbiology
Stages of biofilm development during biofilm-related infections. (a) Biofilm formation begins with planktonic bacteria attaching to host tissue. (b) Initial adhesion may take place under body fluid flow or no-flow conditions, depending on the tissue and site of adherence. During initial adhesion, bacteria sense the surface, which alters their physiology and can prepare them for a transition to a sessile lifestyle. (c) Cell division can generate daughter cells with uneven distribution of intracellular factors, which can result in cell commitment to a biofilm lifestyle or reversal to the planktonic state. (d) As bacteria multiply and form microcolonies, they also start secreting autoinducers (AI). However, AI are washed away at this stage due to fluid flow in the host and consequently, quorum sensing (QS) is not switched on. (e) Once biofilms start maturing, the production of extracellular polymeric substances (EPS) will retain AI within the biofilm, which will activate QS. (f) Eventually, bacteria in mature biofilms can disperse as planktonic bacteria and colonize other locations. Current Opinion in Microbiology 2020, 53:9–18
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Table 1 Examples of bacterial surface sensing factors in mechanotransduction Bacterial Species
Surface sensor
Reference
E. coli
Type 1 pili/FimH adhesin P pili Outer membrane lipoprotein NlpE & Cpx two-component signal transduction pathway Flagellar basal body protein FliL Flagellar motor MotAB Wsp surface sensing system Type 4 pili Cell wall
[37,44,46] [39] [41]
P. mirabilis P. aeruginosa
S. aureus
promotes the expression of Type 1 pili [46]. FimH binding to mannose-coated beads upregulates transcription of fimB. Recombinase FimB promotes the Phase ON orientation of the fimS invertible element, which encodes the fim operon promoter, and enables the expression of Type I pili to further enhance adhesion [48]. Surface sensing in P. aeruginosa is mediated by Type 4 pili (T4P), the flagellar motor MotAB and the Wsp surface sensing system, triggering an intracellular cyclic diguanylate monophosphate (c-di-GMP) increase in a subpopulation of initially attached cells [49,50,51]. This heterogeneous increase results in a high c-di-GMP population that will commit to a sessile lifestyle and a low c-di-GMP population that will commit to surface motility and dispersion, enabling P. aeruginosa to efficiently colonize the host and spread the infection. The heterogeneous c-di-GMP increase might be the result of Wsp system localization on the lateral side of bacteria and the ability of P. aeruginosa to attach either laterally or polarly. Thus, the Wsp system is activated only on laterally attached cells [51], where it mediates phosphorylation of the WspR di-guanylate cyclase, thereby promoting c-di-GMP synthesis [42]. Increased c-di-GMP levels promote adhesion by binding to the cytoplasmic FimW protein, which then rapidly localizes at the cell poles to promote T4P formation [49]. Asymmetric division of high c-di-GMP cells can generate further heterogeneity by producing daughter cells with an uneven distribution of Pch phosphodiesterase (Figure 1c) [49]. This results in a low c-di-GMP flagellated cell that will resume the planktonic lifestyle and a high c-di-GMP piliated cell that will remain attached. In addition to c-di-GMP, surface sensing also regulates cyclic adenosine monophosphate (cAMP) in P. aeruginosa. Mechanical changes in T4P during adhesion are detected by the Pil-Chp chemosensory system, regulating cAMP production and activating cAMP-dependent processes that control adhesion, biofilm formation, and pathogenicity [45]. cAMP also helps P. aeruginosa ‘remember’ surface adhesion, increasing its readiness to re-attach in a subsequent surface encounter [52]. In surface-sentient cells, adhesion memory is imprinted as coupled out-of-phase oscillations of intracellular cAMP levels and T4P activity, which are transferrable to daughter cells. www.sciencedirect.com
[40] [49] [42,51] [45,50,52,62] [47]
By contrast, cAMP levels and T4P activity are not correlated in surface-naı¨ve cells. Overall, these examples show how initial adhesion and surface sensing can trigger intracellular changes to promote a sessile lifestyle. It remains to be determined whether E. faecalis also possesses surface sensing mechanisms to determine the fate of attached bacteria.
The effect of fluid flow, adhesion, and tissue topography on quorum sensing Bacteria in biofilms coordinate group behavior using quorum sensing (QS), an interbacterial communication system based on the production, sensing, and response to extracellular signaling molecules called autoinducers (AI) [53]. In E. faecalis, three QS systems have been identified: the cytolysin, LuxS and Fsr systems [54]. The cytolysin QS system regulates the expression of the pore-forming toxin cytolysin, whereas a role in biofilm formation has been shown for the Fsr and LuxS systems [54]. The significance of the LuxS system in E. faecalis, however, is not yet well-studied. In the case of the Fsr regulatory system, the FsrD propeptide is processed by FsrB to generate the gelatinase biosynthesis-activating pheromone (GBAP), a small lactone that acts as the AI [54]. The membrane sensor kinase FsrC senses the accumulation of GBAP and phosphorylates the response regulator FsrA, which induces expression of gelatinase (GelE) and serine protease (SprE) [54,55,56]. These proteases control autolysin (Atn)-mediated fratricide and extracellular DNA (eDNA) release, which contributes to biofilm formation in vitro [57]. Using a catheter-associated urinary tract infection (CAUTI) murine model, however, E. faecalis established infection and formed biofilm on catheters even in the absence of atn and gelE [58]. GelE is also involved in fibrin degradation and cleavage of Ace, which mediates adhesion to collagen [59,60]. In line with these roles, infection by an E. faecalis gelE mutant in a rabbit model of endocarditis resulted in increased deposition of a fibrinous matrix layer, which suggested that GelE might be involved in bacterial dissemination by Current Opinion in Microbiology 2020, 53:9–18
12 Host-microbe interactions: bacterial
promoting degradation of the vegetation substrate [61]. Despite current knowledge of Fsr-mediated GelE upregulation during biofilm formation and its downstream roles, little is known about the stimuli that induce Fsr QS in the host. Recent studies in P. aeruginosa and S. aureus have shown how surface adhesion and fluid flow can affect QS. Surfaceassociated P. aeruginosa is more sensitive to QS compared to planktonic cells [62]. Surface association sensed by T4P retraction motors and minor pilins induces the sRNA Lrs1, which in turn induces the QS master regulator LasR. Higher levels of LasR in attached cells in the presence of autoinducer N-(3-oxododecanoyl)-homoserine lactone result in increased transcription of downstream QS target genes compared to planktonic P. aeruginosa [62]. The Agr QS system in S. aureus has an inhibitory effect on biofilm formation, favoring biofilm dispersion instead [63]. A recent study found that exposure of S. aureus to fluid flow can reverse this effect by washing away the endogenously produced AIs [64]. A S. aureus strain harboring an AIresponsive agr P3-gfpmut2 transcriptional fusion exhibited fluorescence in the absence but not in the presence of fluid flow during an 8-h exposure. Addition of exogenous AI under flow conditions restored fluorescence, showing that the endogenously produced AIs were washed away by the flow (Figure 1d). During a 16-h exposure to flow, the fluorescence reporter remained inactive in S. aureus cells at the biofilm-fluid interface and as a result biofilm grew thicker. At the same time, cells at the biofilm base started becoming fluorescent, suggesting that the biofilm mass increase protected AI in the inner layers from being washed away (Figure 1e). This population distribution may be beneficial, as Agr-off bacteria exposed to flow can form thicker and more robust biofilms, whereas an Agr-on population in the biofilm base can potentially contribute to dispersal and dissemination (Figure 1f). The same study also showed how complex topography, such as crevices, creates a microenvironment protected from fluid flow to promote AI accumulation and QS activation in S. aureus. A similar effect of fluid flow on QS activity was shown in P. aeruginosa, where increasing the flow rate resulted in a delayed onset of QS signaling [65]. Interestingly, Psl polysaccharide production, which is QS-independent, precedes the onset of QS, suggesting that early extracellular polymeric substance (EPS) production might prevent autoinducer molecules from being washed away. Overall, these findings show how physiology and tissue geometry in the host, as well as the biofilm matrix, are important parameters for QS activity in biofilms. Since E. faecalis also establishes biofilms in the dynamic environments of the heart and the catheterized urinary bladder, we speculate that the degree of exposure to blood and urine flow in combination with surface association and matrix composition may result in a heterogeneous pattern of Fsr activity in the biofilm, similar to what was described for S. aureus and P. aeruginosa. Current Opinion in Microbiology 2020, 53:9–18
Biofilm strategies to overcome innate host immune responses Biofilm-related infections are often chronic, in part due to the inability of the host to clear biofilm [66]. The expression of biofilm-specific immunomodulatory factors, the presence of EPS, and the large size of biofilms together pose a challenge for successful clearance by immune cells. In E. faecalis, among the factors that compromise immune clearance [67], one is also associated with biofilm-formation. GelE, which is regulated by the Fsr QS, facilitates complement resistance by cleaving complement component C5a in vivo in an infective endocarditis model, thus decreasing neutrophil recruitment [61]. Additionally, GelE cleaves complement component C3, which decreases opsonization and phagocytosis in vitro [68]. Independent of its effects on the
Figure 2
(a)
Degrading enzymes ROS Proteases Phagocytosis
Bacteria Neutrophils
Degranulation
Phagolysozyme
50 μm (b)
NETosis Phagocytosis inhibited EPS 50 μm Current Opinion in Microbiology
Evasion of host immune responses in biofilms. (a) As a response to bacterial infection, neutrophils can employ multiple antimicrobial strategies including phagocytosis and subsequent killing in the phagolysosome via degradative enzymes, as well as degranulation, resulting in the release of reactive oxygen species (ROS) and proteases which are microbicidal. Bacteria that escape these antimicrobial strategies can eventually attach to biotic surfaces and initiate biofilm formation. (b) Biofilm aggregates can be resistant to phagocytosis due to their large physical size and extracellular polymeric substances (EPS). Unable to phagocytose biofilms, neutrophils release neutrophil extracellular traps (NETs). However, the role of NETosis remains ambiguous for biofilms, as it selectively helps confine biofilms of some microorganism to the site of infection, yet contributes to biofilm formation of others [87]. www.sciencedirect.com
Biofilm pathogenesis in vivo Tan, Antypas and Kline 13
immune response, GelE also contributes to biofilm formation by regulating Atn-mediated eDNA release [69,70]. These findings together suggest that GelE may play a bifunctional role in biofilm-associated infection, compromising immune clearance while simultaneously enabling E. faecalis biofilm formation. Biofilm-associated factors that compromise complement components have also been described for P. aeruginosa. In P. aeruginosa, LasB elastase can degrade both C1q and C3 complement components [71], but it is also a critical biofilm factor. Deletion of lasB [72] or treatment with LasB inhibitor, N-mercaptoacetyl-Phe-Tyr-amide [73], leads to reduced biofilm formation in vitro in the absence of host cells. Additionally, LasB elastase stimulates exopolysaccharide alginate biosynthesis, which contributes to both structure and antibiotic resistance in P. aeruginosa biofilms [74].
Phagocytosis is critical for innate immune clearance of pathogens. However, biofilm aggregates are usually much larger in size (4 200 mm) [75], compared to planktonic bacteria (1 3 mm), rendering biofilm aggregates resistant to phagocytosis (Figure 2) [76–81]. Moreover, biofilms are encased in a matrix of EPS that may alter the viscoelastic properties of biofilm in a way that it is refractory to phagocytosis [82]. Enterococcal CAUTI and infective endocarditis are good examples of biofilm-related infections that elicit a strong neutrophil response, yet neutrophils alone cannot clear bacteria without concomitant antibiotic treatment [83,84]. Neutrophils in E. faecalis vegetations in a rat endocarditis model release neutrophil extracellular traps (NETs) [83]. However, their effect of NETs on the outcome of the infection, as well as what triggers their release, has not been reported (Figure 2). In
Figure 3
(a)
(b)
Low iron conditions L-ornithine
E. faecalis
Siderophores
E. coli E. coli E. faecalis
Macrophages
(d)
(c)
Phanto-Valentine leucocidin α-hemolysin Methicillin-resistant S. aureus
Siderophores HQNO Rhamnolipids Alginate P. aeruginosa
P. aeruginosa
S. aureus
Current Opinion in Microbiology
Polymicrobial interactions in biofilm-related infections. Interspecies interactions occur during (a) catheter-associated urinary tract infections (CAUTI), (b and c) wound and (d) lung infections. (a) E. faecalis suppresses the host immune response to promote E. coli in CAUTI. (b) E. faecalis exports a cue to increase siderophore expression in E. coli, which promotes E. coli biofilm formation and virulence during wound infections. (c) P. aeruginosa induces expression of virulence factors in methicillin-resistant S. aureus, which leads to delayed wound re-epithelization and healing. (d) Overproduction of alginate by P. aeruginosa reduces production of virulence factors necessary for killing of S. aureus, which promotes S. aureus colonization in lungs of cystic fibrosis patients. www.sciencedirect.com
Current Opinion in Microbiology 2020, 53:9–18
14 Host-microbe interactions: bacterial
Streptococcus mutans and S. aureus endocarditis vegetations, the release of NETs enhanced biofilm formation and expansion, instead of exerting antimicrobial activity [85,86]. Moreover, P. aeruginosa biofilms formed on the cornea of mice are too large to be phagocytized, and neutrophils are unable to penetrate the biofilm, giving rise to an acellular, protective, ‘dead zone’ composed of NETs between the biofilm and neutrophils [87]. The P. aeruginosa cytotoxin ExoS is required for NET release and formation of the dead zone. The P. aeruginosa biofilm EPS component Psl also prevents neutrophil infiltration and promotes the dead zone. Blocking production of both ExoS and Psl results in a switch from neutrophil NET formation to phagocytosis and bacterial killing [87]. A size-dependent NETosis has also been shown for Candida albicans and Mycobacterium bovis, where hyphae and bacterial aggregates but not small yeast or single bacteria triggered NETs release [88]. Although the size and EPS composition of E. faecalis biofilms in vivo have not been sufficiently characterized, it is tempting to speculate that they could play an important role in blocking neutrophils from clearing an enterococcal infection.
Microbe–microbe interactions during infections Interspecies microbe–microbe interactions during biofilmrelated infections can synergistically enhance pathogenesis. E. coli, co-isolated with E. faecalis during CAUTI [89] and wound infection [90,91], derives a benefit and undergoes augmented biofilm formation in the presence of E. faecalis in both CAUTI and wound infection model systems [92,93]. E. faecalis suppresses NF-kB-driven responses in macrophages locally, enabling commensal E. coli to colonize and thus promoting polymicrobial CAUTI (Figure 3a) [93]. Through a different mechanism, export of L-ornithine by E. faecalis signals biosynthesis of the siderophore enterobactin in E. coli to promote E. coli biofilm growth under ironlimiting conditions and in polymicrobial wound infections (Figure 3b) [92]. Enterobactin is crucial in promoting biofilm development, maturation, and survival in E. coli [94], demonstrating how one species can promote the expression of a factor in another species that enables biofilm formation and consequently increases infection severity. In another example, mixed but not single species biofilms of methicillin resistant S. aureus and P. aeruginosa significantly downregulated keratinocyte growth factor 1 (KGF1), resulting in delayed re-epithelization in a porcine wound healing model [95]. In the same model, P. aeruginosa also induced the expression of staphylococcal virulence factors PantonValentine eucocidin and a-hemolysin, increasing the severity of infection (Figure 3c) [95]. S. aureus and P. aeruginosa are also often co-isolated from the lungs of cystic fibrosis (CF) patients. Although P. aeruginosa isolated from monoinfected CF patients outcompetes S. aureus in vitro, P. aeruginosa isolates from co-infected CF patients are more permissive to S. aureus growth [96]. The ability of S. aureus to grow in Current Opinion in Microbiology 2020, 53:9–18
co-infections is due to the overproduction of alginate in P. aeruginosa strains derived from CF co-infections, which in turn reduces the production of factors necessary for robust killing of S. aureus, enabling S. aureus to establish biofilmassociated infections (Figure 3d) [96]. As E. faecalis can often be co-isolated with S. aureus or P. aeruginosa during wound infections [6,97], examining these interspecies interactions will also be important for understanding how these polymicrobial infections initiate and persist.
Conclusions In this review, we selected recent concepts in biofilm pathogenesis from different pathogens and presented them through the lens of E. faecalis biofilm-related infections to define outstanding questions in enterococcal biofilm infection biology. These concepts highlight how mechanical cues from the host and cooperation with other bacterial species alter bacterial physiology and generate a phenotypically heterogeneous population of biofilm-associated and planktonic bacteria to guarantee survival against the immune system and infection dissemination. Lessons learned from these studies demonstrate the importance of investigating how E. faecalis responds to fluid flow and surface adhesion and how these cues might trigger physiological changes to direct the transition between planktonic and biofilm state. Exploring the EPS composition, as well as the biofilm interplay with immune cells, will also help us to understand why E. faecalis biofilms are not cleared by the immune system. Given that E. faecalis is often co-isolated with other pathogens, it will be important to mechanistically understand whether and how interspecies interactions affect pathogenesis. Characterization of host– biofilm interactions with spatiotemporal and single-cell resolution using new in vitro microfluidic models, together with animal models and omics technologies, will help us open the black box of enterococcal biofilm pathogenesis and ultimately design new diagnostic and treatment strategies.
Conflict of interest Nothing to declare.
CRediT authorship contribution statement Casandra Ai Zhu Tan: Conceptualization, Writing original draft, Writing - review & editing, Visualization. Haris Antypas: Conceptualization, Writing - original draft, Writing - review & editing. Kimberly A Kline: Conceptualization, Writing - review & editing, Supervision, Funding acquisition.
Acknowledgements Work in the Kline laboratory related to this article is supported by the Ministry of Education Singapore under its Research Centre of Excellence Program and by the Ministry of Education Singapore under its Tier 2 programme (MOE2018-T2-1-127) and the National Medical Research Council (OFIRG18may-0039). Preparation of this review article was also financially supported by Knut and Alice Wallenberg Foundation under its Wallenberg-NTU postdoctoral fellowship programme (to H.A.) and the Interdisciplinary Graduate Programme of Nanyang Technological University (to C.A.Z.T.). Figures in this article are created with BioRender. com. www.sciencedirect.com
Biofilm pathogenesis in vivo Tan, Antypas and Kline 15
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Gambino M, Cappitelli F: Mini-review: biofilm responses to oxidative stress. Biofouling 2016, 32:167-178.
2.
Kavanaugh JS, Horswill AR: Impact of environmental cues on Staphylococcal quorum sensing and biofilm development. J Biol Chem 2016, 291:12556-12564.
3.
Tolker-Nielsen T: Biofilm development. Microbiol Spectr 2015, 3 MB-0001-2014.
4.
Toyofuku M, Inaba T, Kiyokawa T, Obana N, Yawata Y, Nomura N: Environmental factors that shape biofilm formation. Biosci Biotechnol Biochem 2016, 80:7-12.
5.
Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK: Antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006–2007. Infect Control Hosp Epidemiol 2008, 29:996-1011.
6.
Gjødsbøl K, Christensen JJ, Karlsmark T, Jørgensen B, Klein BM, Krogfelt KA: Multiple bacterial species reside in chronic wounds: a longitudinal study. Int Wound J 2006, 3:225-231.
7.
Peng D, Li X, Liu P, Luo M, Chen S, Su K, Zhang Z, He Q, Qiu J, Li Y: Epidemiology of pathogens and antimicrobial resistance of catheter-associated urinary tract infections in intensive care units: a systematic review and meta-analysis. Am J Infect Control 2018, 46:e81-e90.
8.
Agudelo Higuita NI, Huycke MM: Enterococcal disease, epidemiology, and implications for treatment. In Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Edited by Gilmore MS, Clewell DB, Ike Y, Shankar N. Massachusetts Eye and Ear Infirmary; 2014.
9.
Tonkin ML, Boulanger MJ: The shear stress of host cell invasion: exploring the role of biomolecular complexes. PLoS Pathog 2015, 11:e1004539.
10. Ch’ng J-H, Chong KK, Lam LN, Wong JJ, Kline KA: Biofilm associated infection by enterococci. Nat Rev Microbiol 2019, 17:82-94 A comprehensive review of E. faecalis and E. faecium pathogenesis with a focus on biofilm-related infections, persistence, polymicrobial biofilms and novel treatment strategies. 11. Beaussart A, Baker AE, Kuchma SL, El-Kirat-Chatel S, O’Toole GA, Dufreˆne YF: Nanoscale adhesion forces of Pseudomonas aeruginosa type IV pili. ACS Nano 2014, 8:1072310733. 12. Claes J, Liesenborghs L, Peetermans M, Veloso T, Missiakas D, Schneewind O, Mancini S, Entenza J, Hoylaerts M, Heying R: Clumping factor A, von Willebrand factor-binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall. J Thromb Haemost 2017, 15:1009-1019. 13. Herman-Bausier P, Labate C, Towell AM, Derclaye S, Geoghegan JA, Dufreˆne YF: Staphylococcus aureus clumping factor A is a force-sensitive molecular switch that activates bacterial adhesion. Proc Natl Acad Sci U S A 2018, 115:55645569 The authors elucidated the mechanistic details underlying staphylococcal ClfA adhesion to fibrinogen, showing a force-activated mechanism similar to the E. coli catch bond, which explains how S. aureus adheres and causes an infection to the host under high shear stress. 14. Mairey E, Genovesio A, Donnadieu E, Bernard C, Jaubert F, Pinard E, Seylaz J, Olivo-Marin J-C, Nassif X, Dume´nil G: Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood–brain barrier. J Exp Med 2006, 203:1939-1950. 15. Melican K, Sandoval RM, Kader A, Josefsson L, Tanner GA, Molitoris BA, Richter-Dahlfors A: Uropathogenic Escherichia coli P and Type 1 fimbriae act in synergy in a living host to facilitate www.sciencedirect.com
renal colonization leading to nephron obstruction. PLoS Pathog 2011, 7:e1001298. 16. Pappelbaum KI, Gorzelanny C, Gra¨ssle S, Suckau J, Laschke MW, Bischoff M, Bauer C, Schorpp-Kistner M, Weidenmaier C, Schneppenheim R: Ultralarge von Willebrand factor fibers mediate luminal Staphylococcus aureus adhesion to an intact endothelial cell layer under shear stress. Circulation 2013, 128:50-59. 17. Thomas WE, Nilsson LM, Forero M, Sokurenko EV, Vogel V: Shear-dependent ‘stick-and-roll’ adhesion of type 1 fimbriated Escherichia coli. Mol Microbiol 2004, 53:1545-1557. 18. Nallapareddy SR, Sillanpa¨a¨ J, Mitchell J, Singh KV, Chowdhury SA, Weinstock GM, Sullam PM, Murray BE: Conservation of Ebp-type pilus genes among Enterococci and demonstration of their role in adherence of Enterococcus faecalis to human platelets. Infect Immun 2011, 79:2911-2920. 19. Nallapareddy SR, Singh KV, Sillanpa¨a¨ J, Zhao M, Murray BE: Relative contributions of Ebp pili and the collagen adhesin Ace to host extracellular matrix protein adherence and experimental urinary tract infection by Enterococcus faecalis OG1RF. Infect Immun 2011, 79:2901-2910. 20. Nielsen HV, Guiton PS, Kline KA, Port GC, Pinkner JS, Neiers F, Normark S, Henriques-Normark B, Caparon MG, Hultgren SJ: The metal ion-dependent adhesion site motif of the Enterococcus faecalis EbpA pilin mediates pilus function in catheterassociated urinary tract infection. mBio 2012, 3 e00177-12. 21. Singh KV, Nallapareddy SR, Murray BE: Importance of the ebp (endocarditisand biofilm-associated pilus) locus in the pathogenesis of Enterococcus faecalis ascending urinary tract infection. J Infect Dis 2007, 195:1671-1677. 22. Flores-Mireles AL, Walker JN, Bauman TM, Potretzke AM, Schreiber HL, Park AM, Pinkner JS, Caparon MG, Hultgren SJ, Desai A: Fibrinogen release and deposition on urinary catheters placed during urological procedures. J Urol 2016, 196:416-421. 23. Nallapareddy SR, Qin X, Weinstock GM, Ho¨o¨k M, Murray BE: Enterococcus faecalis adhesin, ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I. Infect Immun 2000, 68:5218-5224. 24. Rozdzinski E, Marre R, Susa M, Wirth R, Muscholl-Silberhorn A: Aggregation substance-mediated adherence of Enterococcus faecalis to immobilized extracellular matrix proteins. Microb Pathog 2001, 30:211-220. 25. Lebreton F, Riboulet-Bisson E, Serror P, Sanguinetti M, Posteraro B, Torelli R, Hartke A, Auffray Y, Giard J-C: ace, which encodes an adhesin in Enterococcus faecalis, is regulated by Ers and is involved in virulence. Infect Immun 2009, 77:28322839. 26. Singh KV, Nallapareddy SR, Sillanpa¨a¨ J, Murray BE: Importance of the collagen adhesin Ace in pathogenesis and protection against Enterococcus faecalis experimental endocarditis. PLoS Pathog 2010, 6:e1000716. 27. Shankar N, Lockatell CV, Baghdayan AS, Drachenberg C, Gilmore MS, Johnson DE: Role of Enterococcus faecalis surface protein ESP in the pathogenesis of ascending urinary tract infection. Infect Immun 2001, 69:4366-4372. 28. Fabretti F, Theilacker C, Baldassarri L, Kaczynski Z, Kropec A, Holst O, Huebner J: Alanine esters of Enterococcal lipoteichoic acid play a role in biofilm formation and resistance to antimicrobial peptides. Infect Immun 2006, 74:4164-4171. 29. Forero M, Yakovenko O, Sokurenko EV, Thomas WE, Vogel V: Uncoiling mechanics of Escherichia coli type I fimbriae are optimized for catch bonds. PLoS Biol 2006, 4:e298. 30. Lecuyer S, Rusconi R, Shen Y, Forsyth A, Vlamakis H, Kolter R, Stone HA: Shear stress increases the residence time of adhesion of Pseudomonas aeruginosa. Biophys J 2011, 100:341-350. 31. Thomas W, Forero M, Yakovenko O, Nilsson L, Vicini P, Sokurenko E, Vogel V: Catch-bond model derived from allostery Current Opinion in Microbiology 2020, 53:9–18
16 Host-microbe interactions: bacterial
explains force-activated bacterial adhesion. Biophys J 2006, 90:753-764. 32. Weaver WM, Dharmaraja S, Milisavljevic V, Di Carlo D: The effects of shear stress on isolated receptor–ligand interactions of Staphylococcus epidermidis and human plasma fibrinogen using molecularly patterned microfluidics. Lab Chip 2011, 11:883-889. 33. Kerrigan SW, Loughmann A, Meade G, Foster TJ, Cox D: Staphylococcus aureus clumping factor mediates rapid thrombus formation under high shear. Blood 2006, 108:1816. 34. Harapanahalli AK, Younes JA, Allan E, van der Mei HC, Busscher HJ: Chemical signals and mechanosensing in bacterial responses to their environment. PLoS Pathog 2015, 11:e1005057. 35. Persat A: Bacterial mechanotransduction. Curr Opin Microbiol 2017, 36:1-6. 36. Stones DH, Krachler AM: Against the tide: the role of bacterial adhesion in host colonization. Biochem Soc Trans 2016, 44:1571-1580. 37. Otto K, Norbeck J, Larsson T, Karlsson K-A, Hermansson M: Adhesion of type 1-fimbriated Escherichia coli to abiotic surfaces leads to altered composition of outer membrane proteins. J Bacteriol 2001, 183:2445-2453. 38. Lele PP, Hosu BG, Berg HC: Dynamics of mechanosensing in the bacterial flagellar motor. Proc Natl Acad Sci U S A 2013, 110:11839-11844. 39. Zhang JP, Normark S: Induction of gene expression in Escherichia coli after pilus-mediated adherence. Science 1996, 273:1234-1236. 40. Lee Y-Y, Belas R: Loss of FliL alters Proteus mirabilis surface sensing and temperature-dependent swarming. J Bacteriol 2015, 197:159-173. 41. Otto K, Silhavy TJ: Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc Natl Acad Sci U S A 2002, 99:2287-2292. 42. O’Connor JR, Kuwada NJ, Huangyutitham V, Wiggins PA, Harwood CS: Surface sensing and lateral subcellular localization of WspA, the receptor in a chemosensory-like system leading to c-di-GMP production. Mol Microbiol 2012, 86:720-729. 43. Kansal R, Rasko DA, Sahl JW, Munson GP, Roy K, Luo Q, Sheikh A, Kuhne KJ, Fleckenstein JM: Transcriptional modulation of enterotoxigenic Escherichia coli virulence genes in response to epithelial cell interactions. Infect Immun 2013, 81:259-270.
Pseudomonas aeruginosa. Cell Host Microbe 2019, 25:140-152. e6 The authors showed how the flagellar motor mediates surface sensing in P. aeruginosa, triggering polar localization of FimW bound by c-di-GMP and in turn leading to Type 4 pili assembly and enhanced adhesion. Adhesion is followed by an asymmetric division, which generates two daughter cells with high and low levels of c-di-GMP that gave rise to a sessile and a planktonic cell respectively. This single-cell study shows how surface sensing can alter physiology and prepare bacteria for a sessile lifestyle. Moreover, it demonstrates the phenomenon of bacterial labour division, which serves as an efficient strategy for uniform host colonization. 50. Rodesney CA, Roman B, Dhamani N, Cooley BJ, Katira P, Touhami A, Gordon VD: Mechanosensing of shear by Pseudomonas aeruginosa leads to increased levels of the cyclic-di-GMP signal initiating biofilm development. Proc Natl Acad Sci U S A 2017, 114:5906-5911 This article showed how Pel polysaccharide production and shear stress increases friction between attached P. aeruginosa and a surface and leads to an increase in c-di-GMP intracellular levels. This type of surface sensing requires fully functional Type 4 pili. 51. Armbruster CR, Lee CK, Parker-Gilham J, de Anda J, Xia A, Zhao K, Murakami K, Tseng BS, Hoffman LR, Jin F: Heterogeneity in surface sensing suggests a division of labor in Pseudomonas aeruginosa populations. eLife 2019, 8:e45084 The authors studied c-di-GMP production in single surface-associated P. aeruginosa and identified a high c-di-GMP subpopulation committed to biofilm formation and a low c-di-GMP subpopulation committed to surface motility. This heterogeneity was linked to the different activity levels of the Wsp surface sensing system among attached bacteria. 52. Lee CK, de Anda J, Baker AE, Bennett RR, Luo Y, Lee EY, Keefe JA, Helali JS, Ma J, Zhao K: Multigenerational memory and adaptive adhesion in early bacterial biofilm communities. Proc Natl Acad Sci U S A 2018, 115:4471-4476 The authors identified a “learning-by-doing” mechanism in P. aeruginosa, in which bacteria remember surface adhesion events over several generations, increasing their readiness to re-attach compared to surfacenaı¨ve cells. This memory is based on coupled out-of-phase oscillations of cAMP levels and Type 4 pili activity. 53. Rutherford ST, Bassler BL: Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med 2012, 2:a012427. 54. Ali L, Goraya MU, Arafat Y, Ajmal M, Chen J-L, Yu D: Molecular mechanism of quorum-sensing in Enterococcus faecalis: its role in virulence and therapeutic approaches. Int J Mol Sci 2017, 18(5):960 In this review, recent literature about the cytolysin, LuxS, Fsr quorum sensing systems is discussed, as well as their role in enterococcal infections (a comprehensive table is included with relevant references), and potential therapeutic strategies.
44. Bhomkar P, Materi W, Semenchenko V, Wishart DS: Transcriptional response of E. coli upon FimH-mediated fimbrial adhesion. Gene Regul Syst Biol 2010, 4:1-17.
55. Qin X, Singh KV, Weinstock GM, Murray BE: Characterization of fsr, a regulator controlling expression of gelatinase and serine protease in Enterococcus faecalis OG1RF. J Bacteriol 2001, 183:3372-3382.
45. Persat A, Inclan YF, Engel JN, Stone HA, Gitai Z: Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2015, 112:7563-7568.
56. Qin X, Singh KV, Weinstock GM, Murray BE: Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence. Infect Immun 2000, 68:2579-2586.
46. Schwan WR, Beck MT, Hung CS, Hultgren SJ: Differential regulation of Escherichia coli fim genes following binding to mannose receptors. J Pathog 2018, 2018:2897581 This study shows how the FimH-mannose interaction upregulates transcription of recombinase fimB and downregulates transcription of recombinase fimE, which shifted the fimS promoter to phase ON and upregulated expression of Type I pili.
57. Thomas VC, Hiromasa Y, Harms N, Thurlow L, Tomich J, Hancock LE: A fratricidal mechanism is responsible for eDNA release and contributes to biofilm development of Enterococcus faecalis. Mol Microbiol 2009, 72:1022-1036.
47. Harapanahalli AK, Chen Y, Li J, Busscher HJ, van der Mei HC: Influence of adhesion force on icaA and cidA gene expression and production of matrix components in Staphylococcus aureus biofilms. Appl Environ Microbiol 2015, 81:3369-3378.
58. Guiton PS, Hung CS, Hancock LE, Caparon MG, Hultgren SJ: Enterococcal biofilm formation and virulence in an optimized murine model of foreign body-associated urinary tract infections. Infect Immun 2010, 78:4166-4175.
48. Schwan WR: Regulation of fim genes in uropathogenic Escherichia coli. World J Clin Infect Dis 2011, 1:17-25.
59. Pinkston KL, Gao P, Diaz-Garcia D, Sillanpa¨a¨ J, Nallapareddy SR, Murray BE, Harvey BR: The Fsr quorum-sensing system of Enterococcus faecalis modulates surface display of the collagen-binding MSCRAMM Ace through regulation of gelE. J Bacteriol 2011, 193:4317-4325.
49. Laventie B-J, Sangermani M, Estermann F, Manfredi P, Planes R, Hug I, Jaeger T, Meunier E, Broz P, Jenal U: A surface-induced asymmetric program promotes tissue colonization by
60. Waters CM, Antiporta MH, Murray BE, Dunny GM: Role of the Enterococcus faecalis GelE protease in determination of cellular chain length, supernatant pheromone levels, and
Current Opinion in Microbiology 2020, 53:9–18
www.sciencedirect.com
Biofilm pathogenesis in vivo Tan, Antypas and Kline 17
degradation of fibrin and misfolded surface proteins. J Bacteriol 2003, 185:3613-3623.
phagocytosis of Streptococcus pneumoniae. Infect Immun 2013, 81:2606-2615.
61. Thurlow LR, Thomas VC, Narayanan S, Olson S, Fleming SD, Hancock LE: Gelatinase contributes to the pathogenesis of endocarditis caused by Enterococcus faecalis. Infect Immun 2010, 78:4936-4943.
78. Horvath DJ Jr, Li B, Casper T, Partida-Sanchez S, Hunstad DA, Hultgren SJ, Justice SS: Morphological plasticity promotes resistance to phagocyte killing of uropathogenic Escherichia coli. Microbes Infect 2011, 13:426-437.
62. Chuang SK, Vrla GD, Fro¨hlich KS, Gitai Z: Surface association sensitizes Pseudomonas aeruginosa to quorum sensing. Nat Commun 2019, 10:1-10 This study shows how surface association can lead to the upregulation of quorum sensing master regulator LasR via sRNA Lrs1. Consequently, surface-associated cells can achieve higher levels of quorum sensing activity compared to planktonic cells in the presence of autoinducer N-(3oxododecanoyl)-homoserine lactone.
79. Jesaitis AJ, Franklin MJ, Berglund D, Sasaki M, Lord CI, Bleazard JB, Duffy JE, Beyenal H, Lewandowski Z: Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions. J Immunol 2003, 171:4329-4339.
63. Boles BR, Horswill AR: Agr-mediated dispersal of Staphylococcus aureus biofilms. PLoS Pathog 2008, 4: e1000052.
80. Thurlow LR, Hanke ML, Fritz T, Angle A, Aldrich A, Williams SH, Engebretsen IL, Bayles KW, Horswill AR, Kielian T: Staphylococcus aureus biofilms prevent macrophage phagocytosis and attenuate inflammation in vivo. J Immunol 2011, 186:6585-6596.
64. Kim MK, Ingremeau F, Zhao A, Bassler BL, Stone HA: Local and global consequences of flow on bacterial quorum sensing. Nat Microbiol 2016, 1:15005.
81. van Gennip M, Christensen LD, Alhede M, Qvortrup K, Jensen PØ, Høiby N, Givskov M, Bjarnsholt T: Interactions between polymorphonuclear leukocytes and Pseudomonas aeruginosa biofilms on silicone implants in vivo. Infect Immun 2012, 80:2601-2607.
65. Emge P, Moeller J, Jang H, Rusconi R, Yawata Y, Stocker R, Vogel V: Resilience of bacterial quorum sensing against fluid flow. Sci Rep 2016, 6:33115.
82. Stewart PS: Biophysics of biofilm infection. Pathog Dis 2014, 70:212-218.
66. Bjarnsholt T: The role of bacterial biofilms in chronic infections. APMIS Suppl 2013, 121:1-58.
83. Augustin P, Alsalih G, Launey Y, Delbosc S, Louedec L, Ollivier V, Chau F, Montravers P, Duval X, Michel JB: Predominant role of host proteases in myocardial damage associated with infectious endocarditis induced by Enterococcus faecalis in a rat model. Infect Immun 2013, 81:1721-1729.
67. Kao PHN, Kline KA: Jekyll and Mr. Hide: how Enterococcus faecalis subverts the host immune response to cause infection. J Mol Biol 2019, 431:2932-2945 An extensive review on how E. faecalis transits from a commensal bacterium to a pathogen and the strategies it adopts to suppress or evade the host innate and adaptive immune responses elicited during infections.
84. Rousseau M, Goh HS, Holec S, Albert ML, Williams RB, Ingersoll MA, Kline KA: Bladder catheterization increases susceptibility to infection that can be prevented by prophylactic antibiotic treatment. JCI Insight 2016, 1:e88178.
68. Park SY, Shin YP, Kim CH, Park HJ, Seong YS, Kim BS, Seo SJ, Lee IH: Immune evasion of Enterococcus faecalis by an extracellular gelatinase that cleaves C3 and iC3b. J Immunol 2008, 181:6328-6336.
85. Jung CJ, Yeh CY, Hsu RB, Lee CM, Shun CT, Chia JS: Endocarditis pathogen promotes vegetation formation by inducing intravascular neutrophil extracellular traps through activated platelets. Circulation 2015, 131:571-581.
69. Kristich CJ, Li Y-H, Cvitkovitch DG, Dunny GM: Esp-independent biofilm formation by Enterococcus faecalis. J Bacteriol 2004, 186:154-163.
86. Hsu CC, Hsu RB, Ohniwa RL, Chen JW, Yuan CT, Chia JS, Jung CJ: Neutrophil extracellular traps enhance Staphylococcus aureus vegetation formation through interaction with platelets in infective endocarditis. Thromb Haemost 2019, 119:786-796.
70. Thomas VC, Thurlow LR, Boyle D, Hancock LE: Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development. J Bacteriol 2008, 190:5690-5698. 71. Hong Y, Ghebrehiwet B: Effect of Pseudomonas aeruginosa elastase and alkaline protease on serum complement and isolated components C1q and C3. Clin Immunol Immunopathol 1992, 62:133-138. 72. Yu H, He X, Xie W, Xiong J, Sheng H, Guo S, Huang C, Zhang D, Zhang K: Elastase LasB of Pseudomonas aeruginosa promotes biofilm formation partly through rhamnolipidmediated regulation. Can J Microbiol 2014, 60:227-235. 73. Cathcart GR, Quinn D, Greer B, Harriott P, Lynas JF, Gilmore BF, Walker B: Novel inhibitors of the Pseudomonas aeruginosa virulence factor LasB: a potential therapeutic approach for the attenuation of virulence mechanisms in pseudomonal infection. Antimicrob Agents Chemother 2011, 55:2670-2678. 74. Hentzer M, Teitzel GM, Balzer GJ, Heydorn A, Molin S, Givskov M, Parsek MR: Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J Bacteriol 2001, 183:5395-5401. 75. Bjarnsholt T, Alhede M, Alhede M, Eickhardt-Sørensen SR, Moser C, Ku¨hl M, Jensen PØ, Høiby N: The in vivo biofilm. Trends Microbiol 2013, 21:466-474. 76. Bernut A, Herrmann J-L, Kissa K, Dubremetz J-F, Gaillard J-L, Lutfalla G, Kremer L: Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation. Proc Natl Acad Sci U S A 2014, 111:E943-E952. 77. Domenech M, Ramos-Sevillano E, Garcı´a E, Moscoso M, Yuste J: Biofilm formation avoids complement immunity and www.sciencedirect.com
87. Thanabalasuriar A, Scott BNV, Peiseler M, Willson ME, Zeng Z, Warrener P, Keller AE, Surewaard BGJ, Dozier EA, Korhonen JT: Neutrophil extracellular traps confine Pseudomonas aeruginosa ocular biofilms and restrict brain invasion. Cell Host Microbe 2019, 25:526-536.e4 The authors investigated P. aeruginosa pathogenesis in keratitis and found that neutrophils release NETs at the base of the biofilms and is dependent on the T3SS of P. aeruginosa, which in turn prevents dissemination of P. aeruginosa into the brain. Blocking both T3SS PcrV and exopolysaccharide Psl with antibodies enables neutrophils to break down biofilms by transiting from NETosis to intracellular phagocytosis and killing. 88. Branzk N, Lubojemska A, Hardison SE, Wang Q, Gutierrez MG, Brown GD, Papayannopoulos V: Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol 2014, 15:1017-1025. 89. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ: Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 2015, 13:269-284. 90. Citron DM, Goldstein EJ, Merriam CV, Lipsky BA, Abramson MA: Bacteriology of moderate-to-severe diabetic foot infections and in vitro activity of antimicrobial agents. J Clin Microbiol 2007, 45:2819-2828. 91. Giacometti A, Cirioni O, Schimizzi A, Del Prete M, Barchiesi F, D’errico M, Petrelli E, Scalise G: Epidemiology and microbiology of surgical wound infections. J Clin Microbiol 2000, 38:918-922. 92. Keogh D, Tay W, Ho Y, Dale J, Chen S, Umashankar S, Williams RH, Chen S, Dunny G, Kline K: Enterococcal metabolite cues facilitate interspecies niche modulation and polymicrobial infection. Cell Host Microbe 2016, 20:493-503. Current Opinion in Microbiology 2020, 53:9–18
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93. Tien BYQ, Goh HMS, Chong KKL, Bhaduri-Tagore S, Holec S, Dress R, Ginhoux F, Ingersoll MA, Williams RB, Kline KA: Enterococcus faecalis promotes innate immune suppression and polymicrobial catheter-associated urinary tract infection. Infect Immun 2017, 85:e00378-00317 This paper shows that E. faecalis suppresses macrophage activation via NF-kB, which in turn promotes commensal E. coli strain colonization in a polymicrobial CAUTI model.
Pseudomonas aeruginosa in polymicrobial wound infection. PLoS One 2013, 8:e56846.
94. May T, Okabe S: Enterobactin is required for biofilm development in reduced-genome Escherichia coli. Environ Microbiol 2011, 13:3149-3162.
96. Limoli DH, Whitfield GB, Kitao T, Ivey ML, Davis MR, Grahl N, Hogan DA, Rahme LG, Howell PL, O’Toole GA: Pseudomonas aeruginosa alginate overproduction promotes coexistence with Staphylococcus aureus in a model of cystic fibrosis respiratory infection. mBio 2017, 8 e00186-00117 The authors found that P. aeruginosa with a mucoid phenotype due to overproduction of alginate, reduces the production of virulence factors which are needed for killing of S. aureus and hence enabling the coexistence between the two species in a cystic fibrosis respiratory infection model.
95. Pastar I, Nusbaum AG, Gil J, Patel SB, Chen J, Valdes J, Stojadinovic O, Plano LR, Tomic-Canic M, Davis SC: Interactions of methicillin resistant Staphylococcus aureus USA300 and
97. James GA, Swogger E, Wolcott R, Pulcini Ed, Secor P, Sestrich J, Costerton JW, Stewart PS: Biofilms in chronic wounds. Wound Repair Regen 2008, 16:37-44.
Current Opinion in Microbiology 2020, 53:9–18
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