Heterogeneity governs 3D-cultures of clinically relevant microbial communities

doi:10.21203/rs.3.rs-2715275/v1

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Heterogeneity governs 3D-cultures of clinically relevant microbial communities

https://doi.org/10.21203/rs.3.rs-2715275/v1

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published 21 Aug, 2023

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A distinctive feature of the biological substrates hosting bacterial niches is their intrinsic heterogeneity, which should be retained in in vitro cultures to closely represent the complex microbial ecology. Here, we design and characterise 3D substrates providing bacteria with environments that possess structural heterogeneity and spontaneous microscopic dynamics. As a case study, we generate by diffusion-induced gelation a mucin-based hydrogel (CF-Mu³Gel) bioinspired on cystic fibrosis (CF) mucus, a microbial niche challenging current therapeutic strategies. We demonstrate that gradients in the properties of the CF-Mu³Gel impact the organisation and the antimicrobial tolerance in mono- and co-cultures of S. aureus and P. aeruginosa. This leads to typical microbial aggregates and generates anoxic regions mimicking CF clinical features that standard cultures are unable to emulate. Our findings shed new light on the understanding of how the substrate influences microbial behaviour, providing a new platform to develop novel effective and possibly personalised therapies.

Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance

Health sciences/Medical research/Preclinical research

Health sciences/Diseases/Infectious diseases/Bacterial infection

Health sciences/Medical research/Drug development

Biological sciences/Biological techniques/Biological models/Respiratory system models

Structural, rheological, and compositional heterogeneity is a pervasive feature of most bacteria-hosting habitats. It has a significant impact on microbial organisation, metabolic activity and adopted pathways, and is further linked with energy consumption, gene expression and biomass production.¹ Within these complex 3D environments, individual bacteria are subjected to time-varying spatial gradients of nutrients, metabolites and oxygen tension, all of which act as the driving forces of chemotaxis and aerotaxis. In multicellular communities, collective microbial dynamics contribute further to the heterogeneity of the environmental niche, generating a complex ecosystem characterised by both microbe-microbe and microbe-environment interaction dynamics. These features must somehow be rendered in in vitro models, and they are not reproducible with cultures in intrinsically homogeneous liquid media (planktonic conditions).

Cystic fibrosis (CF) mucus is a challenging example of a heterogeneous microbial ecosystem. The presence of Staphylococcus aureus and Pseudomonas aeruginosa in CF mucus is generally associated with diminished lung function and more rapid pulmonary decline, but there is no consensus as to whether these bacteria are antagonistic or if S. aureus and P. aeruginosa are able to coexist in a complex microenvironment. Some studies indicate that early colonisation by P. aeruginosa shows strong antagonism towards S. aureus,^2,3 through the secretion of a variety of anti-staphylococcal molecules and proteases that inhibit S. aureus growth and proliferation.^4,5 This process induces a metabolic transition of S. aureus from aerobic respiration to fermentation and eventually leads to a loss of S. aureus viability.⁶ In response to this hostile environment, S. aureus may adapt to P. aeruginosa exoproducts by increasing biofilm formation and the presence of small colony variants.^5,7 Yet, other studies show that S. aureus supports colonisation and pathogenicity of P. aeruginosa,⁸ and that anaerobiosis is required for their coexistence.⁹

Crucially, novel models are needed to address the contribution of the environment in bacterial cultures and co-cultures, in particular to enhance knowledge about the mechanisms of bacterial colonisation and how bacteria-bacteria and bacteria-environment interaction dynamics unfold in complex microbial ecosystems. Such models can help to screen existing antimicrobial agents and to support the development of new, patient-specific, molecules which are possibly closer to the actual clinical infections context, tackling infections occurring in mucus.¹⁰In vitro models exhibiting the key characteristics of the mucus environment and that, at the same time, provide crucial information on pharmacokinetics in a high throughput fashion, would allow for a more effective screening of the most promising compounds early on in the drug discovery phase. These will simultaneously reduce the number of animals used during pre-clinical analyses.¹¹ Existing in vitro models range from traditional methods, such as microtiter plates and dynamic systems,¹² to 3D printing of bacteria.^13,14 Co-cultures of S. aureus and P. aeruginosa have been performed using modified media in static¹⁵ or dynamic conditions,^{9, 16–19} anoxia, microtiter plates,^20,21 Calgary biofilm device²² or even directly over mammalian cells.^6,23 The co-culture of these bacteria is of extreme importance, not only because of their mutual impact on behaviour and metabolic activity, but also because some studies have shown that their interplay contributes to antimicrobial tolerance.^18,19,24,25 The challenge however, is that these existing models, despite shining some light on the behaviour of these bacteria in 3D conditions, these systems lack the heterogeneous environment that bacteria experience in the CF airway niche.

In this paper, we exploit a new approach based on a controlled diffusion of a crosslinking agent to generate a 3D structure that has the advantage of retaining spatial gradients, permitting a microscopic-restructuring dynamics of the microstructure, and allowing for spatial control of oxygen content. This enabled the development of an in vitro model, which is bioinspired to a specific microbiological environment, in this case the CF airway mucus, and that supports the study of the bacterial interplay within a heterogeneous microbial ecosystem.

Extracting the key features of mucus to reproduce a heterogeneous bacterial environment. CF mucus models, CF-Mu³Gel, were synthesised by slow calcium permeation through a membrane to slowly produce a 3D heterogeneous structure. CF-Mu³Gel is predominantly composed of mucin and sodium chloride, whose ranges are within those reported in the literature for CF sputum.^26,27 CF-Mu³Gel was produced using two common bacterial culture media, Müller Hinton broth (MHB) or Luria Bertani broth (LB). This reproduces the complex 3D mucus environment while retaining the fundamental composition of the culture media. Viscoelastic properties of the hydrogels were not strongly affected by the different culture media, although CF-Mu³Gel produced in MHB displayed closer characteristics to those of CF mucus reported in the literature (Fig. 1a and b).²⁸ As CF mucus characteristics vary, not only between patients, but also within a single patient in a short period of time,^29–33 the concentration of Ca²⁺ ions was varied to produce CF-Mu³Gel with viscoelastic properties (Supplementary Fig. 1a and b) tailorable to the desired stiffness according to disease progression³⁴ or even patient-specific to support personalised pharmacological treatment. The average mesh size, estimated for all tested CF-Mu³Gel formulations, was similar independently of the composition. However, the CF-Mu³Gel produced in MHB exhibited a closer mesh size to that reported for CF sputum than those produced in LB and NaCl (Fig. 1c).²⁹ As a result, all further analyses were therefore performed on CF-Mu³Gel produced in MHB (Fig. 1d), as this maximised the representation of CF mucus structural properties.

Gradients have a tremendous impact on microbial behaviour and though they are present in the majority of human mucus, the methods to characterise them are scarce. Photon Correlation Imaging (PCI) enabled the visualisation of both gradients in the structure and microscopic dynamics of the CF-Mu³Gel to be detected via scattered intensity and correlation maps, respectively. The correlation maps evidence that diffusion-controlled gelation produced hydrogels which were stiffer and almost dynamically arrested in the regions first exposed to Ca²⁺ ions (orange region, top map in Fig. 1e). The zones far from the diffusion inlet are more tenuous and display high degrees of spontaneous restructuring dynamics (green regions). Besides, CF-Mu³Gel retains a gradual reduction of O₂ tension from top to its bottom, decreasing from 280 to 194 µmol.l^− 1 at y = 0 to 2300 µm, respectively (Fig. 1f, 1g).

Structural and O₂ gradients affect the topographical organisation of S. aureus and P. aeruginosa. CF-Mu³Gel kept its integrity up to the end of the test (48h) when cultured with S. aureus and P. aeruginosa. After 72h of incubation in different media, sterile CF-Mu³Gel retained its dimensions with variations of 20% (weight) and 40% (thickness) on average when incubated in both isotonic and hypertonic media, and with variations of 44% (weight) and 200% (thickness) when incubated in hypotonic medium alone (Supplementary Fig. 1d and e).

Mono-cultures of S. aureus and P. aeruginosa in CF-Mu³Gel and planktonic conditions reached the stationary phase at 24h with counts of approximately 10⁹ CFU/ml (Fig. 2a and b). It is notable that an interlaboratory study showed a similar number for both bacteria after 24h of culture, demonstrating that the results obtained in CF-Mu³Gel are laboratory- and operator-independent (Supplementary Fig. 2).

O₂ profiles across CF-Mu³Gel indicated that, independently of the microorganism, O₂ content decreased in relation to the depth and extent to which the CF-Mu³Gel was colonised, even after 12h of culture (Fig. 2d and e). This decrease was more pronounced for longer periods of incubation, and O₂ tension was progressively reduced to a completely anoxic zone, at approximately 300 µm of depth, after 48h of culture (Fig. 2d and e).

During the culture period, the number of S. aureus within CF-Mu³Gel increased and exhibited depth-dependent differences in growth and in the presence of bacterial aggregates, these were visible after 24h culture (Fig. 2a; Supplementary Fig. 3) and increased in size with the progression of the time of culture (Supplementary Fig. 3). Regardless of the observed position, a longer culture time resulted in a higher number of P. aeruginosa within the CF-Mu³Gel (Fig. 2b, Supplementary Fig. 4). P. aeruginosa was able to migrate and colonise the whole thickness of CF-Mu³Gel, with a higher number of bacteria at the top and bottom of CF-Mu³Gel.

Bacteria in CF-Mu³Gel resist antimicrobial treatment. The Minimal Inhibitory Concentration (MIC) of different antimicrobial agents (ciprofloxacin, tobramycin and colistin), which are commonly administered to CF patients with S. aureus and/or P. aeruginosa infections was assessed according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (Supplementary Fig. 6). The determined MIC values were the starting point to further treat bacteria incubated in CF-Mu³Gel for 24h.

A significant antimicrobial effect was observed when S. aureus was cultured under planktonic conditions (four-log₁₀ and six-log₁₀ decline of colonies with 1 and 10 MIC, respectively; Fig. 3a and b). The antimicrobial treatment was less effective towards S. aureus cultured in CF-Mu³Gel at short treatment times (two-log₁₀ and four-log₁₀ decline for 1 and 10 MIC, respectively; Fig. 3a). When bacteria were cultured for extended periods, to simulate established infection conditions prior to antimicrobial treatment, the number of S. aureus colonies in CF-Mu³Gel remained constant, even when a high concentration of ciprofloxacin was used (10 MIC; Fig. 3b). In contrast, a more pronounced antimicrobial effect was observed in planktonic S. aureus, with a decline in colonies of two-log₁₀ and three-log₁₀ with 1 and 10 MIC, respectively, when compared with bacteria grown without antimicrobial treatment (Fig. 3a).

P. aeruginosa was more resistant to antimicrobial treatment when cultured within CF-Mu³Gel than in a liquid medium (Fig. 3c-e). In fact, in planktonic cultures, the number of CFU decreased progressively with increasing concentrations of both ciprofloxacin and tobramycin, resulting in a steep reduction of six-log₁₀ and nine-log₁₀ after 24h of treatment with 1 and 10 MIC, respectively, when compared with the untreated control group (Fig. 3c and d). In contrast, the concentration of 10 MIC tobramycin was found to be three times less effective against P. aeruginosa in CF-Mu³Gel than in planktonic conditions (Fig. 3d). Additionally, colistin treatment was found to be ineffective against bacteria grown within CF-Mu³Gel, even at high concentrations, as it was not able to eradicate P. aeruginosa (Fig. 3d), while bacteria cultured under planktonic conditions were only susceptible to 10 MIC of colistin.

CF-Mu³Gel governs the permeability of antimicrobial agents. CF-Mu³Gel was coupled with either Transwell® supports or PAMPA (parallel artificial membrane permeability assays) membranes, widely used during permeability assessment³⁶ to understand the role of CF-Mu³Gel on antimicrobial retention and consequent antimicrobial susceptibility. CF-Mu³Gel strongly hindered the passage of the three antimicrobial agents (Fig. 3f-h), independently of the supporting system. The differences between Transwell® supports and PAMPA were observable but were irrelevant when the effect of CF-Mu³Gel is taken into account (Fig. 3f-h).

S. aureus and P. aeruginosa co-exist and compete in CF-Mu³Gel in different culture set-ups. Microbial ecosystems composed of S. aureus and P. aeruginosa coexisted in vitro in CF-Mu³Gel and influenced the growth of each other (Fig. 4a-c) and their topographical organisation (Fig. 4d-f), independently of culture method. As the timing of colonisation may affect the way they interact, three different types of co-culture were simulated: (1) a contemporary co-culture of both S. aureus and P. aeruginosa for 24h (Fig. 4a and d); (2) a culture with S. aureus first, followed by a P. aeruginosa culture for another 24h of incubation (Fig. 4b and e); and finally (3) a culture with P. aeruginosa first, after which S. aureus was added and incubated for further 24h (Fig. 4c and f). Inoculation of a contemporary co-culture provides both pathogens with equal conditions for these to thrive (Fig. 4a). After 24h of co-culture, both bacteria were present in CF-Mu³Gel (Fig. 4a), and these were able to colonise the 3D structure (Fig. 4d). This was not observed in planktonic cultures, as S. aureus was not able to grow.

To reproduce CF airways microbial colonisation timeline,³⁸ we began by first culturing S. aureus (experimental setting 2). After 48h of culture, both S. aureus and P. aeruginosa co-existed either in CF-Mu³Gel or planktonic conditions (Fig. 4b). The relative abundance of S. aureus in respect to P. aeruginosa was negligible in planktonic conditions (Fig. 4b).

When P. aeruginosa was first cultured in CF-Mu³Gel, experimental setting (3), both S. aureus and P. aeruginosa co-existed within CF-Mu³Gel, though the relative abundance of S. aureus was minor (Fig. 4c). Under planktonic conditions, P. aeruginosa outcompeted S. aureus (Fig. 4c).

The topographical organisation in co-cultures indicated that both S. aureus and P. aeruginosa formed bacterial aggregates (Fig. 4d-f). This was also observed in mono-cultures of S. aureus and P. aeruginosa within CF-Mu³Gel (Supplementary Fig. 3–5). However, the aggregates of P. aeruginosa produced in experimental setup (3) (Fig. 4f) were larger than those observed in experimental setting (2) (Fig. 4e), possibly due to the longer culture time of P. aeruginosa.

When S. aureus was cultured first, mimicking the microbial colonisation timeline of CF airways³⁸, experimental setting (2), the co-existence of the two bacteria impacted their susceptibility to antimicrobial treatment. In CF-Mu³Gel, no differences were observed in the number of both S. aureus and P. aeruginosa were found when these were co-cultured among the tested ciprofloxacin concentrations (Fig. 5b and d). Interestingly, the bacterial survival rates in CF-Mu³Gel of both S. aureus and P. aeruginosa after ciprofloxacin treatment were higher when in co-cultures than they were in mono-cultures. The opposite was found to be the case in S. aureus co-cultured in planktonic conditions (Fig. 5a).

CF-Mu³Gel supports the growth of mono- and poly-microbial communities. It does so by replicating key features of the microbial ecology within the human mucus in terms of 3D-structural gradients, physical and chemical composition, oxygen distribution, and its ability to function as a barrier to antimicrobial diffusion, antagonistic phenomena and bacteria susceptibility to antimicrobials.

Our intention was to model the airway CF mucus by replicating the viscoelastic properties and mucin content, which has previously been reported for airway CF mucus.^26,28,39 The viscoelastic properties of CF-Mu³Gel increased with frequency (Fig. 1a and b). This has been linked to limited mucus clearance creating an environment for infections to thrive.^3,24,40 The gradient structure of CF-Mu³Gel (Fig. 1e and f) is similar to the structure of CF airway mucus, where stagnating mucins form a higher number of entanglements at the lower strata of the mucus at the airway-epithelium interface. The gelation, induced by diffusion of the crosslinking agent, results in reproducible gradients of structural stiffness, spontaneous restructuring power, and receptiveness to oxygen.

Oxygen gradients within native microbial ecosystems are known to affect bacterial metabolic activity and influence their susceptibility to antimicrobial agents.⁴⁰ The O₂ gradient is a unique feature of CF-Mu³Gel, with a lower O₂ tension at the bottom and a higher O₂ tension, linked to low crosslinking density, at the top (Fig. 1f and g). This profile is related to the diffusion of O₂ in hydrogels, dependent on bound, free, and interfacial water content, mesh size, and crosslinking density.⁴¹ Given that CF-Mu³Gel exhibits a gradient structure with a tighter mesh at the bottom than at the top (Fig. 1e), higher water-to-polymer ratios are found at the top than at the bottom (Fig. 1g). Consequently, higher levels of O₂ are available at the top for bacteria metabolism.

CF-Mu³Gel sustained the growth of S. aureus and P. aeruginosa (Fig. 2a-b, Fig. 3a-e “No ATB”). In CF-Mu³Gel, the P. aeruginosa number was comparable to the numbers found in the lungs of CF patients (10⁸-10¹⁰ bacteria.ml^− 1), while the number of S. aureus was even higher.³⁵S. aureus aggregates were found to increase in number and size over time in CF-Mu³Gel (Fig. 2a, Supplementary Fig. 3). A similar number and type of aggregates of S. aureus formed in a more complex system containing agarose gels.⁴² Though, agarose gels have a uniform structure and rely on bacteria O₂ consumption to produce the gradient.⁴² Whenever bacteria are introduced in CF-Mu³Gel, they are able to sense, self-organise and exacerbate the O₂ profile (Fig. 2d and e). Indeed, the resultant O₂ profile reproduces that determined in CF mucus colonised by S. aureus and P. aeruginosa.^43,44

Agar or alginate 3D systems such as beads,^45–49 and, more recently, a hydrogel composed of alginate/mucin,⁵⁰ have been proposed as suitable means to culture P. aeruginosa. The latter is similar to our previous mucus models^51,52 and all of them lack the presence of 3D gradients. Indeed, our results show that bacteria are able to sense the intrinsic gradients of CF-Mu³Gel. In fact, P. aeruginosa forms multicellular aggregates, similar in morphology and size to those seen in CF mucus and chronic wounds (Fig. 2b; Supplementary Figs. 4 and 5).^53,54 The formation of these aggregates is associated with O₂ gradients,^{43, 55–57} with higher concentration of aggregates at higher O₂ concentrations. This organisation leads to increased production of alginate by P. aeruginosa,^56,58 a process which is believed to be the underlying cause of chronic infections.⁵⁹ This level of organisation was also observed in vitro in alginate beads, in which P. aeruginosa aggregates were mostly present at the surface.^46,47 Supplementation with NO₃⁻ promoted the formation of aggregates in deeper layers. In the CF-Mu³Gel, however, there was no need for NO₃⁻ supplementation to achieve deeper layer colonisation and aggregate formation (Supplementary Fig. 4). The same happened in alginate beads, where steep O₂ gradients were present in CF-Mu³Gel (Fig. 2d-e). Anoxia was found after 48h of culture at 200–300 µm depth with a close similarity to airway CF mucus (Fig. 2d-e).⁴³

Microbial ecology seems to be highly dependent on the presence of a 3D structure and on the culture period. Different culture conditions have been tested to co-culture S. aureus and P. aeruginosa in vitro.^{8,9, 16–22,60–62} Some studies suggest that P. aeruginosa outcompetes S. aureus,^2,3,6 others refer that S. aureus supports colonisation and pathogenicity of P. aeruginosa⁸ or that anaerobiosis is required for their coexistence.⁹ In CF-Mu³Gel, both S. aureus and P. aeruginosa co-existed independently of the experimental condition adopted (Fig. 4), while in planktonic conditions, these only co-existed when S. aureus was cultured first followed by the addition of P. aeruginosa (Fig. 4b). Co-cultures under planktonic conditions, by definition, lack a 3D support, meaning that the exoproducts secreted by P. aeruginosa and other molecules do not freely and easily spread in the medium, thus negatively influencing the survival of S. aureus. Yet, within a 3D substrate, bacteria by-products do slowly diffuse due to interactive and steric phenomena characteristic of 3D environments. This supports the results with CF-Mu³Gel, both S. aureus and P. aeruginosa co-exist with a similar number as in the case with CF mucus (Fig. 4a-c).^35,37 The presence of O₂ gradients may also be a key feature to enable simultaneous growth. Under anoxia, S. aureus growth did not change when co-cultured with either strains or clinical isolates of P. aeruginosa.⁹ In the CF-Mu³Gel, both bacteria were able to colonise and formed aggregates across the 3D structure, which appear in contact with each other (Fig. 4d-f, Supplementary Fig. 7). When the co-culture period was prolonged from 48h (Fig. 4) to 72h (Supplementary Fig. 8 “No ATB”), P. aeruginosa progressively overtook S. aureus. Indeed, the size of P. aeruginosa aggregates increased over longer incubation times, as can be seen in Fig. 4e and f, in which P. aeruginosa was allowed to grow 24h in co-culture with pre-cultured of S. aureus (S. aureus first, followed by another 24h of co-culture with P. aeruginosa), while in the second P. aeruginosa was allowed to grow for full 48h (P. aeruginosa first, followed by another 24h of co-culture with S. aureus). The organisation of P. aeruginosa aggregates (Fig. 4e-f) negatively influenced S. aureus growth (Fig. 4b; Supplementary Fig. 8). This result is supported by previous studies on the P. aeruginosa/S. aureus antagonistic phenomenon, in which co-culture induced the secretion of anti-staphylococcal molecules and proteases.^4,5

It is clearly understood that antimicrobial treatment drastically reduces CF patient’s morbidity and increases life expectancy.^63,64 The key therapeutic challenge, however and as addressed in this paper, is that efficacy testing on planktonically cultured bacteria is misleading.

Indeed, it is estimated that the antimicrobial tolerance of bacteria in biofilms is 100-1,000 times greater than those determined planktonically.⁶⁵ With our approach, we reproduced what occurs in CF antimicrobial treatment conditions. This is evidenced by the fact that in the CF-Mu³Gel the number of bacteria after exposure to the minimal inhibitory concentration (MIC) was three orders of magnitude higher than those found under planktonic conditions. Overall, even at antimicrobial concentrations which are lower or higher than the MIC, S. aureus and P. aeruginosa were more resistant to antimicrobial treatment when cultured within CF-Mu³Gel, independently of the antimicrobial agent (Fig. 3a-e). S. aureus exhibited high tolerance towards ciprofloxacin, even when shorter incubation periods were adopted, an effect previously linked to reduced ATP levels, associated with inhibition of S. aureus respiration by ciprofloxacin.⁶⁶ The CF-Mu³Gel, as CF mucus, provides a 3D steric and interactive barrier to the diffusion of antimicrobial agents, a phenomenon which may explain the lack of efficiency of antimicrobial treatment. Again, the CF-Mu³Gel, as CF mucus together with the exopolysaccharidic matrix produced by bacteria, offered a significant barrier to the diffusion of all antimicrobial agents (Fig. 3f-h). The diffusion of colistin sulphate, tobramycin, and ciprofloxacin was strongly reduced by CF-Mu³Gel, also possibly due to the interaction of these drugs with mucin⁶⁷, its main component. Low permeability through the mucus may limit the amount of drug that comes into contact with both S. aureus and P. aeruginosa, thus affecting its efficacy. In this sense it is significant to note that the MIC of these antimicrobial agents against S. aureus and P. aeruginosa was shown to be one to three orders of magnitude higher in the presence of mucin.^67,68 The lack of antimicrobial efficiency can be also linked to the formation of persister populations.⁶⁹ Co-cultures modified the resistance to ciprofloxacin and in CF-Mu³Gel, both S. aureus and P. aeruginosa were found to be more resistant to ciprofloxacin treatment when co-cultured than when in mono-cultures (Fig. 5b and d). This is supported by the literature, as S. aureus co-cultured with P. aeruginosa revealed to be more resistant to ciprofloxacin treatment when in the presence of one another.^70,71 All of this underscores and emphasises the need for significantly more reliable screening tools.

The distinctive features of CF-Mu³Gel enabled dual-species survival and formation of clinically-witnessed microcolony aggregates which are characteristic of CF mucus and chronic wounds. The interplay of these features resulted in a similar barrier to antimicrobial treatment and, in some cases, we were able to reproduce the mismatch between planktonically cultured bacteria and patients samples. From a technological point of view, CF-Mu³Gel offers enormous versatility and potential as both the composition and viscoelastic properties can be tuned to closely and accurately model different environmental conditions. In the wide library of antimicrobial agents that can be tested, ciprofloxacin, tobramycin and colistin were selected because these are the most commonly administered treatments for CF therapy and because they have distinctive modes of action. Although this suggests that the platform is versatile, we cannot confirm that the observed effects can be extrapolated to other antimicrobial agents. We have in this paper considered the two main pathogens colonising CF airways and have demonstrated the efficacy of this approach. Further studies are needed to investigate and confirm the ability of CF-Mu³Gel to sustain the growth of multiple pathogens, and possibly the CF microbiota retrieved from different patients. Our work has the potential to create opportunities for the development of personalised and effective antimicrobial treatments.

CF-Mu³Gel properties. G-CF-Mu³Gel® (for short, it was termed CF-Mu³Gel) is mostly composed of 2.5% (w/v) porcine stomach type III mucin (Sigma-Aldrich, M1778; lot# SLBQ7188V, Germany) and 0.71% (wt/vol) sodium chloride that falls within the ranges previously determined in CF sputum,^26,27 produced in MHB, LBB or in 7.07mg/ml NaCl exploiting the licensed technology of Bac³Gel, Lda, Portugal.³⁴ Briefly, G-CF-Mu³Gel® was produced through controlled diffusion of different concentrations of Ca²⁺ ions ranging from 0.12–1.20% (w/v) to a mucin-based precursor solution leading to the generation of 3D structures with spatial gradients.

Rheological characterisation. The viscoelastic properties of CF-Mu³Gel produced in different media (7.07 mg.ml^− 1 NaCl, MHB and LB) were evaluated using an Anton Paar MCR502 Rheometer (Austria) with a 25 mm diameter plate geometry (serial number 52530/19910) at 25ºC. The linear viscoelastic region (LVR) was determined through strain sweep analyses employing a strain logarithmic ramp varying from 0.1 to 1000% at a frequency of 1Hz. Oscillatory frequency sweeps were further performed to evaluate both storage, G', and loss, G'', moduli, as well as complex shear modulus, G*, at 0.5% (at strain amplitudes within the linear regime) with frequencies changing logarithmically in the 0.1–20 Hz range. The viscoelastic properties of CF-Mu³Gel were further compared to those reported for CF sputum.²⁸ A detailed analysis was conducted at the characteristic frequencies of breathing (0.5 Hz) and ciliary beating (10 Hz) under physiologic conditions. The rheological data was further exploited to estimate the mesh size of different formulations of CF-Mu³Gel by exploiting both the generalised Maxwell model and the rubber elasticity theory, as previously reported.^34,51

Photon correlation imaging (PCI). PCI is an optical technique that blends the power of Dynamic Light Scattering (DLS) with that of digital imaging. A classical DLS experiment allows one to investigate the microscopic dynamics of a sample by measuring the degree of correlation c(t, t+𝞽) between the intensity of the light scattered by the sample at two different times t and t+𝞽. In the simplest case of the Brownian motion of a dispersed nanoparticle, for instance, this quantity is simply related to particle diffusion over distances of the order of the inverse of the scattered wave vector q = 4𝞹/𝞴 sin (𝞱/2), where 𝞱 is the angle at which the scattered light is measured (in our experiment, 𝞱=90°) and is the wavelength of the scattered light. PCI yields similar information but, by operating on an image of the sample produced on a CMOS multi-pixel camera by a suitable “stopped-down” optics,⁷² retains at the same time the spatial resolution of an imaging system. This allows the spatial distribution of both the scattered intensity and of the degree of correlation at the given delay time t to be mapped (respectively dubbed “intensity” and “correlation” maps). To our aims, PCI has two main advantages with respect to standard DLS:

a) it provides spatial distribution of both the scattered intensity and of the degree of correlation, thus allowing to spot and quantify heterogeneity both in the structure and in the microscopic dynamics of the sample.

b) standard DLS measurements require the medium to be “ergodic”, namely that the scatterers are free to move over distances that are not much smaller than the wavelength. This condition, which is required for the averages in time measured in a DLS experiment to coincide with true statistical averages (“ensemble” averages), is satisfied by Brownian particle dispersions, but not by a hydrogel, where the microscopic motion is almost completely frozen. This limitation is overcome by PCI, where a correct ensemble average is easily obtained from a spatial average over a suitable but still “region of interest”. Thus, PCI provides a unique method to investigate the slow and spatially limited spontaneous restructuring dynamics of soft disordered solids.⁷²

Bacterial strains and culture conditions. The microorganisms used were Staphylococcus aureus ATCC® 25923 (S. aureus) and Pseudomonas aeruginosa ATCC® 15692 (PA01 strain, P. aeruginosa), kindly supplied by R. Migliavacca (Department of Clinical Surgical, Diagnostic and Paediatric Sciences, University of Pavia, Italy). Brain heart infusion broth (BHI; Sigma-Aldrich, 53286, Germany) and Luria Bertani broth (LB; Formedium, LMM0102, United Kingdom) were used to inoculate S. aureus and P. aeruginosa, respectively. Bacteria were grown overnight in their appropriate medium, under aerobic conditions at 37°C using a shaker incubator (VDRL Stirrer 711/CT, Asal Srl, Italy). Both cultures were prepared at the final density of 1×10⁹ bacteria.ml^− 1 as determined by comparing the optical density at 600 nm (OD600) of the sample with a standard curve relating OD600 to bacterial number by using a UV-VIS spectrophotometer (Aurogene S.r.l., HJ1908003, Italy).⁷³ Müller Hinton broth (MHB; Sigma-Aldrich, 70192, Germany) was used to prepare the final inoculum of both bacterial mono- and co-cultures to be inoculated in CF-Mu³Gel or used as planktonic culture controls. The total number of colony forming units (CFU) was counted by plating the serial dilutions (prepared in 0.9% NaCl) of bacterial cultures on Müller Hinton agar (MH agar; Sigma-Aldrich, 70191, Germany) plates, while cetrimide agar (Sigma-Aldrich, 22470, Germany) and mannitol salt (Sigma-Aldrich, 63567, Germany) were used as selective media to count the CFU of P. aeruginosa and S. aureus after co-culture experiments, respectively. All media and plates of MH, mannitol salt and cetrimide agar were prepared following supplier instructions. Sodium citrate tribasic dihydrate (NaCitrate; Sigma-Aldrich, 71404, Germany) was used as a dissolving agent of CF-Mu³Gel to retrieve bacteria that grew within its 3D structure. Three antimicrobial agents were used, including colistin (Sigma-Aldrich, C4461, Germany), ciprofloxacin (Fresenius Kabi, lot# 15LA518P2, Italy), and tobramycin (Ibi, lot# 118B, Italy).

Mono-cultures in CF-Mu³Gel. S. aureus or P. aeruginosa were cultured in CF-Mu³Gel for 24 and 48 h incubation. After overnight culture, the bacterial suspension was subsequently diluted in fresh medium, until reaching the final concentration of 10⁴ bacterial cells.ml^− 1. Cultures in CF-Mu³Gel were achieved by inoculating on the top 100 µl of each separated bacterial suspension and incubating for 24 and 48 h at 37°C. Planktonic cultures of S. aureus or P. aeruginosa (i.e. bacteria cultured in liquid medium without hydrogels) in 96 multiwell flat tissue culture plates were used as controls of the experiment.

Co-cultures in CF-Mu³Gel. Co-cultures of both S. aureus and P. aeruginosa were induced in CF-Mu³Gel in a proportion of 1:1 following three different co-culture settings: a) simultaneous culture of both P. aeruginosa and S. aureus for 24 h; b) culturing first S. aureus for 24 h followed by P. aeruginosa cultured for another 24 h; and finally, c) colonising CF-Mu³Gel with P. aeruginosa first for 24 h, after which S. aureus was added and incubated for further 24 h. Before adding the second bacterial strain, the excess medium was removed and substituted with 100 µl of 10⁴ bacterial cells.ml^− 1 of the secondly introduced bacterium. Co-cultures of S. aureus and P. aeruginosa in planktonic conditions were also carried out as the controls of the experiment for the three different co-culture settings.

Bacterial viability within CF-Mu³Gel. CF-Mu³Gel was dissolved with 150 µl of 50 mM NaCitrate, pH 7.4, for bacterial viability determination. After 2 min contact time, the suspension was diluted to 10^− 6-10^− 10 in 0.9% NaCl solution, pH 7.4, to perform serial dilution and CFU counting. MH agar plates were incubated overnight at 37ºC. When co-cultures were carried out, the total number of CFU was counted using MH agar plates, while cetrimide or mannitol salt agar plates were used as selective media to count the CFU of P. aeruginosa and S. aureus, respectively. CFUs were then calculated taking into consideration a dilution factor of 2.5, resulting from the addition of 50 mM sodium citrate. Planktonic cultures of S. aureus or P. aeruginosa were used as controls of the experiment.

Oxygen tension measurements. O₂ tension of colonised CF-Mu³Gel was measured using a Clark-type O₂ sensor (OX-25; Unisense, Aarhus N, Denmark), connected to the Unisense microsensor multimeter S/N 8678 (Unisense, Denmark), a high sensitivity pico-ampere four-channel amplifier. Before each measurement, the reference anode and the guard cathode were polarised overnight and calibrated with either water saturated with air or with an anoxic solution of 2% (w/w) sodium hydrosulphite. Once the calibration was carefully made, a low melting point agarose consisting of 2% (w/v) agarose in 0.071% (w/v) NaCl (same concentration as in CF-Mu³Gel) was cast into a petri dish. CF-Mu³Gel colonised with either S. aureus or P. aeruginosa were placed over the agarose layer. Microsensors with a tip diameter of 50 µm (OX-50) were positioned at the air – CF-Mu³Gel interface (“depth zero”) using a motorised micromanipulator (Unisense, Denmark). Measurements were performed at the centre of the hydrogels starting at their surface (0 mm) through their thickness, every 100 µm in triplicate, until the tip completely penetrated the whole structure. The maximum depth reached by the tip, termed end depth, was 2300 µm. Sterile CF-Mu³Gel was used as control of the experiment. The Unisense software SensorTrace automatically converts the signal from partial pressure (O₂ tension) to the equivalent O₂ concentration in µmol.l^− 1.

Bacterial Colonisation within CF-Mu³Gel

Preparation of the DsRed-fluorescent strain of S. aureus ATCC 25923. To obtain a DsRed-fluorescent strain, S. aureus ATCC 25923 was transformed by electroporation with the pCAG-DsRed plasmid DNA following the protocol suggested by Schenk and Laddaga (1992).⁷⁴ Chloramphenicol (CP) was used at 10 µg.ml^− 1 (Sigma-Aldrich, Germany). pCAG-DsRed was a gift from Connie Cepko (Addgene plasmid #11151; http://n2t.net/addgene:11151; RRID:Addgene_11151). Plasmid manipulation was performed according to standard techniques.

Preparation of the GFP-fluorescent strain of P. aeruginosa PA01. To obtain a GFP-fluorescent strain, P. aeruginosa PA01 was transformed by electroporation with the pMF230 plasmid DNA following the protocol reported by Cadoret et al. (2014).⁷⁵ Carbenicillin (CB) was used at 300 µg.ml^− 1. pMF230 was a gift from Michael Franklin (Addgene plasmid #62546; http://n2t.net/addgene:62546; RRID:Addgene_62546). Plasmid manipulation was performed according to standard techniques.

Mono-cultures in CF-Mu³Gel. The viability of S. aureus ATCC 25923 or P. aeruginosa PA01 within CF-Mu³Gel was assessed using the Live/Dead dual staining BacLight® Kit (Live/Dead Bacterial Viability Kit, L-7007, Molecular Probes) according to manufacturer specifications. The BacLight® kit is composed of two fluorophores SYTO9 and propidium iodide (PI) that stain total and dead bacteria, respectively. After 10min incubation with SYTO9, the colonised CF-Mu³Gel was washed three times, followed by a 3 min staining with PI. Viable (green) and non-viable (red) bacteria within CF-Mu³Gel were differentiated by using the dual-channel option of the Leica TCS SP8 confocal microscope. Since the P. aeruginosa strain fluoresces green, only the PI or Hoechst 33342 (Hoechst; Invitrogen, Eugene, Code) staining were used, whereas for S. aureus (fluoresces in red) only the SYTO9 was used.

Co-cultures in CF-Mu³Gel. The experiment was performed using DsRed-S. aureus ATCC 25923 (red fluorescence) and GFP-P. aeruginosa PA01 (green fluorescence) to discriminate between the two bacterial strains. The general total number of these bacteria after co-culture in CF-Mu³Gel was evaluated using the Hoechst 33342 staining. Hoechst 33342 can readily cross cell membranes to stain the DNA (blue) of total bacteria (blue). 50 µl of a mixture of Hoechst 33342 with a final concentration of 1 µg.ml^− 1 was added to each colonised CF-Mu³Gel. After Hoechst staining, DsRed-S. aureus ATCC 25923 appeared purple and the GFP-P. aeruginosa PA01 were blue-green.

Confocal Laser Scanning Microscopy (CLSM). CF-Mu³Gel were seeded with DsRed-expressing S. aureus and/or GFP-expressing P. aeruginosa to analyse bacterial colonisation in mono- and co-cultures using CLSM. In this way, cultures were performed in CF-Mu³Gel following the aforementioned method to induce mono- and co-cultures for 12, 24, and 48 h. After each incubation period, colonised CF-Mu³Gel were observed under a high-resolution spectral confocal-laser microscope (Leica TCS SP8 SMD, Leica Microsystems CMS GmbH, Germany). Colonised CF-Mu³Gel were analysed from the top, bottom, and middle. Analyses were performed at different wavelengths according to fluorochrome and staining, namely 550 (570/650) nm excitation for DsRed-expressing S. aureus, 488 (500/550) nm excitation for both GFP-expressing P. aeruginosa and SYTO9, 535 (570/670) nm excitation for PI, and 405 (420/500) nm excitation for Hoechst 33342. The 3D images obtained were analysed using the LasX 3.7.5 software provided by Leica. In the case of mono-cultures by DsRed-S. aureus ATCC 25923, CF-Mu³Gel was supplemented with 10 µg.ml^− 1 CP, whereas in the case of mono-cultures by GFP-expressing P. aeruginosa PA01, CF-Mu³Gel containing 300 µg.ml^− 1 CB was used. As a control, CF-Mu³Gel without bacteria was observed under confocal microscopy at the aforementioned wavelengths to evaluate the absence of possible autofluorescence effects.

Antimicrobial Susceptibility

Determination of Minimal Inhibitory Concentration. Ciprofloxacin, tobramycin, and colistin were selected as antimicrobial agents to which P. aeruginosa is sensitive to determine antimicrobial susceptibility within CF-Mu³Gel, while ciprofloxacin chosen as antimicrobial agents against S. aureus. The minimal inhibitory concentration (MIC) was determined by the broth microdilution method using MHB inoculated with a standard inoculum according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. In this way, 10³ bacterial cells in MHB were incubated with serial dilutions of 1:2 of each antimicrobial agent (0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 mg.l^− 1) for 24 h at 37ºC. OD600 was measured to determine the values of MIC using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) colorimetric test (Sigma-Aldrich, Germany) according to supplier instructions, which transforms the metabolic products of bacteria into a violet observable precipitate that can be read using a spectrophotometer. The intensity of the violet colour is proportional to the number of viable bacteria. The plate was then read at a wavelength of 570 nm and the background read at 630 nm was subtracted (Clariostar microplate reader, BMG Labtech Clariostar, Germany). MIC was established as the lowest concentration of antimicrobial agents that inhibited bacterial growth.

Antimicrobial susceptibility within CF-Mu³Gel. Mono-cultures of either S. aureus or P. aeruginosa in CF-Mu³Gel were induced by culturing 10³ bacteria to achieve 10⁸ bacteria after 24 h of culture. After this period, the supernatant over the hydrogels was removed and these were washed twice with fresh MHB medium. Then, 100 µl of antimicrobial agent at three different concentrations was added, namely 0.1, 1, and 10 MIC. Antimicrobial treatment was carried out for 24 h under static incubation, at 37ºC, after which CFU count was performed. Similarly, co-cultures of P. aeruginosa and S. aureus in CF-Mu³Gel were treated with ciprofloxacin, given that both bacteria are sensitive to this antimicrobial agent. Both S. aureus and P. aeruginosa were cultured in CF-Mu³Gel in a proportion of 1:1 following the co-culture setting b: culturing S. aureus first followed by P. aeruginosa cultured for another 24 h. Several controls were performed: (I) planktonic cultures after antimicrobial treatment; (II) planktonic cultures without antimicrobial treatment; and (III) non-treated but colonised CF-Mu³Gel. Both MHB medium and CF-Mu³Gel without bacteria and antimicrobial treatment were used as controls of sterility.

Drug diffusion studies

Drug diffusion through CF-Mu³Gel. Drug diffusion studies were conducted using either polycarbonate Transwell® supports (Corning Transwell®, CLS3413, Merck) with a porosity of 0.4 µm and inner diameter equal to 6.5 mm or a 96-well plate permeable support system with a filter plate pre-coated with structured layers of phospholipids (PAMPA; Corning® Gentest™ Pre-coated PAMPA, 353015, USA) with a porosity of 0.45 µm and inner diameter equal to 6.2 mm. Drug diffusion through empty Transwell® inserts and empty PAMPA were performed as controls of the experiment. Both ciprofloxacin, tobramycin, and colistin were tested at a concentration of 500 µM. CF-Mu³Gel was introduced on both platforms with a thickness of approximately 500 µm. Afterwards, the donor compartment was filled with the relevant drug solution (200 µl/well) and 300 µl of phosphate saline buffer was added to the acceptor compartment. The filter plate was then coupled to the receiver plate and incubated at room temperature without agitation for 5 and 24 h. At the end of the incubation period, the plates were separated and the volume of both the donor and the receiver plate was collected for further quantification. Liquid chromatography-mass spectrometry was further carried out to quantify the permeated amount of drugs using their relevant calibration curves. The percentage of drug permeated after 5 and 24 h was calculated as follows:

$$\% drug permeated=\frac{{C}_{t}}{{C}_{0}}\times 100$$

where C_t denotes the concentration of drug released at time t, and C₀ represents the initial concentration introduced on the donor compartment. The apparent permeability coefficient (P_app) was expressed using Eq. 1 derived from Fick’s law⁷⁶ for steady state conditions:

$${P}_{app}=\frac{dQ/dt}{{C}_{0}\times A}$$

where dQ is the quantity of drug expressed as moles permeated into the acceptor compartment at time t (18,000 sec), C₀ is the initial concentration in the donor well, and A is the area of the well membrane (0.3 cm²). The P_app was used as an average of all the measures.

Liquid Chromatography–Mass Spectroscopy. An HPLC-MS using a Varian HPLC equipped with a 410 autosampler and an Ascentis C18 column (10cm x 2.1mm, 3µm). Gradient mobile phases composed of acetonitrile and water with 0.1% formic acid as organic and aqueous phases, respectively, were pumped at a flow rate of 200 µl.min^− 1. A flow of 200 µl.min^− 1 and an injection volume of 10 µl were used. Compounds were detected on a Varian 320 MS TQ Mass Spectrometer equipped with an electrospray ionisation (ESI) source operating in positive mode. The detector was used in multiple reaction monitoring (MRM) mode and the transitions of each drug are reported in Supplementary Table 1.

Statistical Analysis. The results of at least three independent experiments are presented as mean ± standard deviation (SD). Statistical analysis was performed using the t-test student and ANOVA using GraphPad Prism version 8 (GraphPad Software, USA). Significant differences were set for *p < 0.05. n corresponds to the number of distinct samples in which different measurements were performed.

Acknowledgements

D.P.P and P.P. would like to thank Switch2Product (UA.A.RRR.ARICID.SVRA.AUTO.AZ18VARI10) for partially funding the validation of the technology to be employed as airway models. This work was also performed thanks to two grants of the Italian Ministry of Education, University and Research (MIUR) to Politecnico di Milano MIUR PRIN 2017 “Soft Adaptive Networks'' and the Department of Molecular Medicine of the University of Pavia under the initiative “Dipartimenti di Eccellenza (2018–2022)”. We are grateful to P. Vaghi (Centro Grandi Strumenti https://cgs.unipv.it/eng/, University of Pavia, Pavia, Italy) for her technical assistance during CLSM analysis. A special thanks to Scott Burgess (University of Pavia, Pavia, Italy) for correcting the English in the manuscript. The schematic representations included in the manuscript were created with BioRender.com.

Author Contributions

The multidisciplinary approach and collaborations established in this work were managed by P.P. with the conceptualisation and direction of the study supported by S.V. and L.V., which equally contributed to the manuscript. D.P.P. and N.S.V. developed the production method of CF-Mu³Gel, under the supervision of P.P. D.P.P., F.B., N.S.V., and A.Z. established and conducted the microbiological and antimicrobial susceptibility analyses under the supervision of LV. D.P.P., A.Z., G.G., and S.v.U. carried out confocal microscopic investigations and analyses. C.S.B. determined the permeability of the antimicrobial agents under the supervision of S.V. D.P.P. performed the rheological characterisation and further analyses of all variations of CF-Mu³Gel under the supervision of F.B.V. D.P.P. and E.C. established and performed the analyses of O₂ tension. D.P.P., S.v.U. prepared the samples for PCI analysis. V.R., S.B analysed PCI data under the supervision of R.P. D.P.P. wrote the original draft of the manuscript and took care of the revisions of the draft, P.P. contributed to the writing and revisions. All authors discussed the results, reviewed the manuscript, and contributed to its final version.

Competing interests

D.P.P., F.B., N.S.V., S.V., L.V., and P.P. are co-inventors of the patented technology. D.P.P. is co-founder, shareholder, and CTO of Bac3Gel, Lda, a mucus‐based company. S.v.U. is co-founder, shareholder, and CEO of Bac3Gel, Lda. S.V., L.V., and P.P. are co-founders, shareholders, and scientific advisors of Bac3Gel, Lda. All other authors declare no competing interests.

Data availability

All data generated or analysed during this work are included in the final article; further data are available upon request to Paola Petrini.

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Yes there is potential Competing Interest. D.P.P., F.B., N.S.V., S.V., L.V., and P.P. are co-inventors of the patented technology. D.P.P. is co-founder, shareholder, and CTO of Bac3Gel, Lda, a mucus‐based company. S.v.U. is co-founder, shareholder, and CEO of Bac3Gel, Lda. S.V., L.V., and P.P. are co-founders, shareholders, and scientific advisors of Bac3Gel, Lda. All other authors declare no competing interests.

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Heterogeneity governs 3D-cultures of clinically relevant microbial communities

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