Biofilm

Staphylococcus aureus is a major human pathogen. Despite high incidence and morbidity, molecular mechanisms occurring during infection remain largely unknown. Under defined conditions, biofilm formation contributes to the severity of S. aureus related infections. Extracellular DNA (eDNA), a component of biofilm matrix released from apoptotic bacteria, is involved in biofilm structure and stability. In many bacterial biofilms, eDNA originates from cell lysis although eDNA can also be actively secreted or exported by bacterial membrane vesicles. By screening the Nebraska transposon library, we identified rpiRc as a biofilm regulator involved in eDNA regulation. RpiRc is a transcription factor from the pentose phosphate pathway (PPP) whose product is a polysaccharide intercellular adhesin (PIA) precursor. However, rpiRc mutant strain showed neither susceptibility to DispersinB(R) (a commercially available enzyme disrupting PIA biofilms) nor alteration of ica transcription (the operon regulating PIA production). Decreased biofilm formation was linked to Sln, an extracellular compound degrading eDNA in an autolysis independent pathway. Biofilm susceptibility to antibiotics in wt and mutant strains was tested using a similar protocol as the Calgary biofilm device. Involvement of RpiRc in S. aureus virulence was assessed ex vivo by internalization experiments into HEK293 cells and in vivo in a mouse model of subcutaneous catheter infection. While minimum inhibitory concentrations (MICs) of planktonic cells were not affected in the mutant strain, we observed increased biofilm susceptibility to almost all tested antibiotics, regardless of their mode of action. More importantly, the rpiRc mutant showed reduced virulence in both ex vivo and in vivo experiments related to decreased fnbpA-B transcription and eDNA production. RpiRc is an important regulator involved in eDNA degradation inside the matrix of mature PIA independent biofilms. These results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also to S. aureus cell adhesion and virulence during subcutaneous infection.\n\nAuthor summaryBiofilm formation contributes to the severity of Staphylococcus aureus related infections. Biofilm matrix is mainly composed by polysaccharide intercellular adhesion (PIA), proteins and extracellular DNA (eDNA). By screening a mutant library of S. aureus, RpiRc was identified as a new regulator of eDNA dependent biofilm formation. How RpiRc regulates biofilm and its role in S. aureus virulence was studied in four different S. aureus strains. Deletion of RpiRc resulted in a pronounced decreased eDNA dependent biofilm formation, but not PIA dependent biofilm formation. Decreased biofilm formation was not related to increased autolysis, but was linked to extracellular compounds found in the supernatant of mutant biofilms. Sln was identified as one of this compound. RpiRc deletion also decreased biofilm recalcitrance (resistance) to selected antibiotics. Involvement of RpiRc in S. aureus pathogenesis was investigated ex vivo by internalization into HEK293 cells and in vivo in a mouse model of catheter infection. RpiRc deletion resulted in decreased virulence related to decreased expression of surface proteins like the fibronectin binding proteins A and B (FnbpA-B). These results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also to S. aureus cell adhesion and virulence during subcutaneous infection.

Phage therapy has been proposed as a therapeutic alternative to antibiotics for treatment of chronic, biofilm-related P. aeruginosa infections. To get a deeper insight into the complex biofilm-phage interactions, we investigated in the present study the effect of three successive exposures to lytic phages of biofilms formed by the reference strains PAO1 and PA14 as well as of two sequential clinical P. aeruginosa isolates from the sputum of a patient with cystic fibrosis (CF). The Calgary device was employed as biofilm model and the efficacy of phage treatment was evaluated by measurements of the biomass stained with crystal violet (CV) and of the cell density of the biofilm bacterial population (CFU/ml) after each of the three phage exposures. The genetic alterations of P. aeruginosa isolates from biofilms exposed to phages were investigated by whole genome sequencing. We show here that the anti-biofilm efficacy of the phage treatment decreased rapidly with repeated applications of lytic phages on P. aeruginosa strains with different genetic background. Although we observed the maintenance of a small subpopulation of sensitive cells after repeated phage treatments, a fast recruitment of mechanisms involved in the persistence of biofilms to the phage attack occurred, mainly by mutations causing alterations of the phage receptors. However, mutations causing phage tolerant phenotypes such as alginate-hyperproducing mutants were also observed. In conclusion, a decreased anti-biofilm effect occurred after repeated exposure to lytic phages of P. aeruginosa biofilms due to recruitment of different resistance and tolerance mechanisms.

Defined as a pneumonia occurring after more than 48 hours of mechanical ventilation via an endotracheal tube, ventilator-associated pneumonia results from biofilm formation on the indwelling tube, seeding the patients lower airways with pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans. Currently there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates biofilm formation of these pathogens in a host-mimicking environment, and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilator airway mucus (SVAM) medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-SEM were employed to characterise the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication, or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved biofilm eradication of all pathogens. Our in vitro endotracheal tube (IVETT) model informs on fundamental microbiology in the ventilator-associated pneumonia context, and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants. ImportanceThe incidence of ventilator-associated pneumonia in mechanically ventilated patients is between 5-40%, increasing to 50-80% in patients suffering from coronavirus disease 2019 (COVID-19). The mortality rate of ventilator-associated pneumonia patients can reach 45%. Treatment of the endotracheal tube biofilms that cause ventilator-associated pneumonia is extremely challenging, with causative organisms able to persist in endotracheal tube biofilm despite appropriate antimicrobial treatment in 56% of ventilator-associated pneumonia patients. Flawed antimicrobial susceptibility testing often means that ventilator-associated pneumonia pathogens are insufficiently treated, resulting in patients experiencing ventilator-associated pneumonia recurrence. Here we present an in vitro endotracheal tube biofilm model that recapitulates key aspects of endotracheal tube biofilms, including dense biofilm growth and elevated antimicrobial tolerance. Thus our biofilm model can be used as a ventilated airway simulating environment, aiding the development of anti-ventilator-associated pneumonia therapies and antimicrobial endotracheal tubes that can one day improve the clinical outcomes of mechanically ventilated patients.

1Opportunistic, biofilm-forming pathogens such as Pseudomonas aeruginosa can employ an array of strategies to reduce the impact of antibiotics on their survival. The biofilm matrix can prevent antibiotics from reaching bacteria embedded within it; general changes in metabolic activity alter susceptibility to specific drugs dependent on the target; changes in the membrane and the expression of channel or pump proteins embedded within it affect drug uptake and efflux; and production of antibiotic-degrading enzymes can remove the threat. In this study, we report that biofilm-deficient mutants of two well-studied lab strains of P. aeruginosa (PA14 and PAO1) have wild-type (WT) levels of tolerance to colistin and meropenem when allowed to establish mature populations in an ex vivo pig lung model of cystic fibrosis lung infection. The biofilm defects in the mutants were confirmed using SEM, and cryoSEM was used to visualise the hydrated biofilm matrix in the WT. Using RNA sequencing of the PA14 WT and an isogenic mutant lacking the pel polysaccharide, we were able to identify a small number of differences in the responses of the two genotypes to the lung environment and to exposure to sub-bactericidal colistin in the lung model. Notably, there was differential upregulation of the MexXY-OprM and MexEF-OprN multidrug efflux pumps. However, the relative roles of biofilm matrix versus cellular changes in physiology in conferring antibiotic tolerance in this environment remain to be fully elucidated.

2.Quantitative image analysis is central to understanding microbial growth, morphology, and spatial organisation. However, conventional metrics such as mean intensity or object count often do not capture the complex structural heterogeneity and patterning characteristic of microbial colonies and biofilms. To address this limitation, Analysis of Biofilm Complexity in 3D (ABC3D), an open-source Python framework for automated extraction of fractal, textural, and statistical descriptors from volumetric microscopy images, is reported. ABC3D computes a set of parameters including fractal dimension, lacunarity, entropy, grey-level co-occurrence matrix features, and wavelet sub-band energies from three-dimensional (3D) image datasets. ABC3D is demonstrated in macrocolony biofilms formed by cell shape mutants of Escherichia coli, where it is shown that nutrient availability accounts for the majority of structural variance, while cell shape produces additional structural variation that differs between nutrient conditions. ABC3D provides researchers with an accessible, quantitative approach to assessing biofilm morphology in microscopy datasets. SummaryAn open-source, quantitative analysis pipeline is presented that integrates fractal, lacunarity, entropy, texture and wavelet descriptors to characterise colony biofilm architecture in three dimensions. Application to Escherichia coli cell shape mutants demonstrates that macrocolony biofilm architecture is best understood as a coordinated, multiscale phenotype rather than as an aggregate of independent structural metrics. 3. Impact statementBiofilm architecture is pivotal for microbial survival, antimicrobial tolerance, and ecological function but tools to quantify structural organisation in these cell communities remain limited. The commonest metrics describe bulk properties such as width, thickness, or cell number, but they do not capture multiscale spatial heterogeneity. Here, an open-source framework for Analysis of Biofilm Complexity in 3 Dimensions (ABC3D) is reported. This software integrates measurements of fractal geometry, lacunarity, entropy, texture statistics, and wavelet energy. ABC3D is demonstrated in Escherichia coli macrocolony biofilms, where it is shown that nutrient environment has a leading role in determining colony architecture in E. coli biofilms, while cell shape has a lesser but still significant influence on structural variation. The ABC3D pipeline can be applied to any microbial communities imaged by confocal microscopy and other volumetric imaging methods and has the potential to give a deeper understanding of how cells organise in biofilms. 4. Data summaryFull code for ABC3D and data analysis is available at https://github.com/gailmcconnell/ABC3D. Image data are available upon request. The author confirms all supporting data, code and protocols have been provided within the article or through supplementary data files.

The opportunistic pathogen Pseudomonas aeruginosa forms biofilm infections in the lungs of people with the genetic condition cystic fibrosis (CF) that can persist for decades. There are numerous P. aeruginosa lifestyle changes associated with chronic biofilm infection cued by the CF lung environment. These include a loss of virulence, metabolic changes and increased antimicrobial tolerance. We have investigated P. aeruginosa PA14 biofilm infection over 7 d in an ex vivo pig lung (EVPL) model for CF, previously shown to facilitate formation of a clinically relevant P. aeruginosa biofilm structure with expression of key genes comparable to human infection. We have compared P. aeruginosa gene expression between sequential time points: 24 h, 48 h and 7 d post infection, and investigated tolerance to polymyxins. Our results demonstrate that the EVPL model can maintain a P. aeruginosa biofilm population, which exhibits increased antibiotic tolerance, for at least 7 d. Differential expression of antimicrobial resistance-associated genes was not observed, however there was significant upregulation of sulfur metabolism and maintenance of a structured biofilm. Our findings provide further insight into the increased P. aeruginosa antibiotic tolerance during chronic infection of the CF lung, and suggest we can cue aspects of chronic infection in 7 d under the right lab conditions.

Recent mesoscopic characterisation of nutrient-transporting channels in E. coli has allowed the identification and measurement of individual channels in whole mature biofilms. However, their complexity under different physiological and environmental conditions remains unknown. Analysis of confocal micrographs of biofilms formed by cell shape mutants of E. coli shows that channels have a high fractal complexity, regardless of cell phenotype or growth medium. In particular, biofilms formed by the mutant strain {Delta}ompR, which has a wide-cell phenotype, have a higher fractal dimension when grown on rich medium than when grown on minimal medium, with channel complexity affected by glucose and agar concentration in the medium. Osmotic stress leads to a dramatic reduction in {Delta}ompR cell size, but has a limited effect on channel morphology. This work shows that fractal image analysis is a powerful tool to quantify the effect of phenotypic mutations and growth environment on the morphological complexity of internal E. coli biofilm structures. If applied to a wider range of mutant strains, this approach could help elucidate the genetic determinants of channel formation in E. coli biofilms.

The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In the Pseudomonas aeruginosa biofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin of P. aeruginosa cells. However, current data describing the effects of OprF in the P. aeruginosa biofilm is limited. Here we identify a nutrient-dependent effect of OprF in static biofilms, whereby {Delta}oprF cells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate that P. aeruginosa {Delta}oprF biofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of mature P. aeruginosa biofilms by retaining eDNA in the matrix. IMPORTANCEMany pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized. However, the effects of P. aeruginosa matrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here we describe a conditional effect of the abundant matrix protein OprF on late-stage P. aeruginosa biofilms. A {Delta}oprF strain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective {Delta}oprF biofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in mature biofilms.

Non-typeable Haemophilus influenzae (NTHi) is an early pathogen isolated from the lungs of children with cystic fibrosis (CF). However, its role in the progression of CF lung infection is poorly understood. Additionally, whether it forms biofilms in the lungs of people with CF is an open question. The development of synthetic cystic fibrosis sputum media has given key insights into the microbiology of later CF pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, through replicating the chemical composition of CF sputum. However, growth of NTHi in these media has not previously been reported. We show that NTHi grows poorly in three variants of synthetic cystic fibrosis sputum media commonly used to induce in vivo -like growth of P. aeruginosa and S. aureus (SCFM1, SCFM2 and SCFM3). The addition of NAD and hemin to SCFM1 and SCFM2 promoted the planktonic growth and biofilm formation of both laboratory and clinical NTHi isolates, and we were able to develop a modified variant of SCFM2 that allows culture of NTHis. We show that NTHi cannot be identified in an established ex-vivo model of CF infection, which uses SCFM and porcine bronchiolar tissue. This may in part be due to the presence of endogenous bacteria on the pig lung tissue which outcompete NTHi, but the lack of selective agar to isolate NTHi from endogenous bacteria, and the fact that NTHi is an exclusively human pathogen, make it hard to conclude that this is the case. Through spiking modified SCFM2 with filter sterilized lung homogenate, biofilm growth of clinical NTHi isolates was enhanced. Our results highlight that there are crucial components present in the lung tissue which NTHi require for growth, that are not present in any published variant of SCFM from the Palmer et al. 2007 lineage. Our results may inform future modifications to SCFM recipes to truly mimic the environment of CF lung sputum, and thus, to facilitate study of a wide range of CF pathogens. Data SummaryThe authors confirm that all supporting data, code and protocols have been provided within the article or through supplementary data files. All raw data has been uploaded to FigShare (https://doi.org/10.6084/m9.figshare.28175300.v1).

Bacterial cells detached from Staphylococcus epidermidis biofilms are found to release predominantly as small oblate clusters ([~]1.9 {micro}m) in both untreated biofilms and biofilms treated with matrix-targeted disruptors. Quantitative image analysis common to colloidal science was applied to quantitatively evaluate the physical properties of 9,147 bacterial clusters detached from S. epidermidis biofilms with and without targeted disruption of individual matrix components (polysaccharides, proteins, extracellular DNA) or solubilization of the extracellular polymeric substances (EPS). Concentrations of S. epidermidis biofilm-detached cells are highest after matrix-targeted disruption of polysaccharides. K-means clustering, an unsupervised machine learning technique, was used to reveal that S. epidermidis biofilm-detached cells are released in five distinct phenotypes: small oblate, mid-sized oblate, large oblate, small spherical, and mid-sized prolate clusters. S. epidermidis biofilm detached cell clusters are predominantly oblate across three size groups (79.5%), with the small oblate phenotype representing 60.1% of cell clusters that have 3.1 {+/-} 1.2 cells per cluster, Euclidean diameters of 1.9 {+/-} 0.4 {micro}m, anisotropy indices of 0.98 {+/-} 0.05, and asphericities of -1.75 {+/-} 0.31 on average. The proportion of S. epidermidis cell clusters within each biofilm-detached cell phenotype differs between matrix-targeted disruptors. There are also variations in the abundance of S. epidermidis biofilm detached cells after matrix-targeted disruption between growth conditions and strains. Evaluating the physical properties of biofilm-detached cells after matrix-targeted disruption is critical to understanding their translocation in fluid flow and susceptibility to the host immune response as well as in evaluating matrix-targeted disruption for biofilm control.

Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances (EPS) and other components such as proteins. Matrix components can be produced by the microorganisms themselves but can also originate from the environment and then be incorporated into the biofilm. For example, we and our collaborators have recently shown that collagen, a host-produced protein that is abundant in many different infection sites, can be taken up into the matrices of Pseudomonas aeruginosa biofilms, altering biofilm mechanics. In an infection, the biofilm matrix protects bacteria from clearance by the immune system, and some of that protection likely arises from the mechanical properties of the biofilm. P. aeruginosa, Staphylococcus aureus, and Burkholderia pseudomallei are human pathogens notable for forming biofilms in vitro and in vivo in tissues rich in collagen such as lung and skin. Here, we show that the incorporation of Type I collagen into P. aeruginosa, S. aureus, and B. pseudomallei biofilms significantly enhances biofilm elasticity and hinders phagocytosis of biofilm bacteria by human neutrophils. Additionally, enzymatic degradation of collagen using collagenase reverses these effects, increasing biofilm susceptibility to neutrophils. Our findings suggest that host materials play significant roles in stabilizing biofilms and may present promising targets for therapeutic interventions.

Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA. MRSA cells also secrete micrococcal nuclease (Nuc1) which degrades double-stranded DNA. In this study we used a nuc1 mutant strain to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. Biofilm was quantitated in microplates using a crystal violet binding assay. Extracellular DNA (eDNA) was isolated from colony biofilms and analyzed by agarose gel electrophoresis. In some experiments, broth or agar was supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods, transferring the rods to fresh broth, and enumerating CFUs that detached from the rods. Biofilm sloughing was investigated by culturing biofilms in glass tubes perfused with broth and measuring the sizes of the detached cell aggregates. We found that a nuc1 mutant strain produced significantly more biofilm and more eDNA than a wild-type strain in both the absence and presence of sub-MIC amoxicillin. nuc1 mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or a wild-type nuc1 gene on a plasmid. In the sloughing assay, nuc1 mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.

Modulator therapies have improved outcomes for people with Cystic Fibrosis (pwCF), and currently more than 50% of pwCF are over the age of 18. This has resulted in an increased prevalence of atypical pathogens, including non-tuberculous mycobacteria (NTM). CF-isolation rates of NTM and Pseudomonas aeruginosa (Pa) are high, and those co-colonized have worse clinical outcomes. We therefore investigated the behavior of these two organisms in a dual-species biofilm. We found that coculture of Mycobacterium abscessus (MAB) promoted biofilm formation by Pa. Confocal imaging revealed changes in biomass and structural organization of the Pa biofilm during coculture with MAB. DNase treatment slightly decreased dual-species biofilm, but biofilm formation was completely abrogated in Pel- and Psl-deficient mutants of Pa. Moreover, dual-species cultures promoted tolerance of Pa to tobramycin treatment. Overall, our findings highlight an interaction between P. aeruginosa and M. abscessus that may result in bacterial persistence for pwCF during antibiotic therapy.

Bacterial aggregates are observed in both natural and artificial environments. In the context of disease, aggregates have been isolated from both chronic and acute infections. Pseudomonas aeruginosa (Pa) aggregates contribute significantly to chronic infections, particularly in the lungs of people with cystic fibrosis (CF). Unlike the large biofilm structures observed in vitro, Pa in CF sputum forms smaller aggregates ([~]10-1000 cells), and the mechanisms behind their formation remain underexplored. This study aims to identify genes essential and unique to Pa aggregate formation in a synthetic CF sputum media (SCFM2). We cultured Pa strain PAO1 in SCFM2 and LB, both with and without mucin, and used RNA sequencing (RNA-seq) to identify differentially expressed genes. The presence of mucin revealed hundreds of significantly differentially expressed (DE) genes, predominantly downregulated, with 40% encoding hypothetical proteins unique to aggregates. Using high-resolution microscopy, we assessed the ability of mutants to form aggregates and identified 13 that were unable to form WT aggregates. Notably, no mutant exhibited a completely planktonic phenotype. Instead, we identified multiple spatial phenotypes described as normal, entropic, or impaired. Entropic mutants displayed tightly packed, raft-like structures, while impaired mutants had loosely packed cells. Predictive modeling linked the prioritized genes to metabolic shifts, iron acquisition, surface modification, and quorum sensing. Co-culture experiments with wild-type PAO1 revealed further spatial heterogeneity and the ability to rescue some mutant phenotypes, suggesting cooperative interactions during growth. This study enhances our understanding of Pa aggregate biology, specifically the genes and pathways unique to aggregation in CF-like environments. Importantly, it provides insights for developing therapeutic strategies targeting aggregate-specific pathways. ImportanceThis study identifies genes essential for the formation of Pseudomonas aeruginosa (Pa) aggregates in cystic fibrosis (CF) sputum, filling a critical gap in understanding their specific biology. Using a synthetic CF sputum model (SCFM2) and RNA sequencing, 13 key genes were identified, whose disruption led to distinct spatial phenotypes observed through high-resolution microscopy. The addition of wild-type cells either rescued the mutant phenotype or increased spatial heterogeneity, suggesting cooperative interactions are involved in aggregate formation. This research advances our knowledge of Pa aggregate biology, particularly the unique genes and pathways involved in CF-like environments, offering valuable insights for developing targeted therapeutic strategies against aggregate-specific pathways.

Mycobacterium abscessus, an inherently drug-resistant, opportunistic, nontuberculous mycobacterium (NTM) predominantly causes pulmonary infections in immunocompromised patients, notably those with cystic fibrosis. M. abscessus subspecies display distinct colony morphologies (rough and smooth), with the prevalent view that M. abscessus (smooth) is a persistent, biofilm-forming phenotype, whilst M. abscessus (rough) is unable to form biofilms. Biofilm formation contributes to persistent infections and exhibits increased antibiotic resistance. We used the chemical mapping technique, nanoscale secondary ion spectrometry (NanoSIMS), to investigate if variations in the biofilm morphology and antibiotic penetration account for the antibiotic susceptibility amongst M. abscessus subspecies, contributing to increased antimicrobial resistance (AMR) and potentially explaining the protracted treatment duration. The susceptibility to bedaquiline (BDQ) of M. abscessus grown as planktonic bacilli and biofilms was measured. The minimum biofilm eradication concentration (MBEC) of BDQ was 8-16 times higher (2-4{micro}g/ml) compared with the minimum inhibitory concentration (MIC) (0.25{micro}g/ml), indicating reduced efficacy against biofilms. Correlative imaging with electron microscopy revealed that M. abscessus (irrespective of the colony morphotype) formed biofilms and that BDQ treatment influenced biofilm morphology. We determined that M. abscessus morphotypes exhibit differential uptake of the antibiotic BDQ in biofilms. M. abscessus subsp. abscessus (smooth) biofilms exhibited the least uptake of BDQ, whereas M. abscessus subsp. bolletii biofilms showed the greatest antibiotic penetration. NanoSIMS analysis revealed no correlation between antibiotic penetration and drug efficacy within the biofilm. This challenges the previous assumption linking biofilm architecture to drug efficacy. Investigating other biofilm characteristics like antibiotic persistence could lead to enhanced treatment approaches. Significance StatementMycobacterium abscessus is an increasingly prevalent pathogen, most often causing lung infections in immunocompromised individuals. Their distinct morphotypes and biofilm-forming capabilities contribute to persistent infections, rendering them challenging to treat with increased antibiotic resistance. This research demonstrates that the antibiotic, bedaquiline exhibits significantly reduced efficacy against M. abscessus growing as a biofilm compared to planktonic growth, but that the efficiency of antibiotic penetration was not the main explanation for the different susceptibilities of MABC biofilms to treatment.

The challenges of defining the biofilm phenotype has been clear for decades. Many biomarkers for biofilm are known, but methods for identifying these are often invasive and/or complicated. These methods often rely on disrupting the biofilm matrix or examining virulence factors and compounds, which may only be expressed under certain conditions. We used microcalorimetric measurements of metabolic energy release to investigate whether unchallenged, planktonic Pseudomonas aeruginosa displayed differences in metabolism compared to surface-bound and non-attached biofilms. The pattern of energy release observed in the recorded microcalorimetric thermograms clearly depended on growth state, though the total energy expenditure was not different between growth states. To characterize these differences, we developed a classification pipeline utilizing machine learning algorithms to classify growth state, based on the observed patterns of energy release. With this approach, we could with high accuracy detect the growth form of blinded samples. To challenge the algorithm, we attempted to limit the amount of training data. By training the algorithm with only a single data point from each growth form, we obtained a mean accuracy of 90.5% using two principal components. Further validation of the classification pipeline showed that the approach was not limited to P. aeruginosa but could also be used for detection of gram-positive Staphylococcus aureus biofilm. We propose that microcalorimetric measurements, in combination with this new quantitative framework, can be used as a non-invasive biomarker to detect the presence of biofilm. These results could have a significant potential in clinical settings where the detection of biofilms in infections often means a different outcome and treatment regime for the patient.

Bacterial biofilms are involved in chronic infections and confer 10 to 1000 times more resistance to antibiotics, leading to treatment failure and complications. When transitioning from a planktonic lifestyle to biofilms, certain Gram-positive bacteria are likely to modulate several cellular pathways including central carbon metabolism, primary biosynthesis pathways and production of secondary metabolites. These metabolic adaptations might play a crucial role in biofilm formation by Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis. Here, we performed a transcriptomic approach to identify cellular pathways that might be similarly regulated during biofilm formation in these bacteria. Different strains and biofilm-inducing media were used to identify a set of regulated genes that are common and independent of the environment or accessory genomes analysed. The gene set enrichment analysis of the transcriptome of four different strains of Gram-positive bacteria identified biosynthesis of secondary metabolites, biosynthesis of antibiotics and purine biosynthesis as three commonly upregulated pathways in biofilm. Our approach did not highlight downregulated pathways during biofilm formation that were common to S. aureus and E. faecalis. Of the three upregulated pathways, the de novo IMP biosynthesis pathway constitutes a promising target of cellular adaptation during biofilm formation. Gene deletions in this pathway, particularly purN, purL, purQ, purH and purM significantly impaired biofilm formation of S. aureus. ImportanceBiofilms are often involved in nosocomial infections and can cause serious chronic infections if not treated properly. Current anti-biofilm strategies rely on antibiotic usage, but they have a limited impact because of the biofilms intrinsic resistance to drugs. Metabolism remodelling likely plays a central role during biofilm formation, but it is still unclear if these cellular adaptations are shared between strains and species. Using comparative transcriptomics of different strains of Staphylococcus aureus and Enterococcus faecalis, we identified a core of commonly regulated genes during biofilm formation. Interestingly, we observed that the de novo IMP biosynthesis was systematically upregulated during biofilm formation. This pathway could constitute an interesting new anti-biofilm target to increase the host spectrum, drug efficiency and prevent resistance evolution. These results are also relevant to a better understanding of biofilm physiology.

Healthcare-associated infections (HAIs) significantly contribute to the burden of antimicrobial resistance (AMR). A major factor in HAIs is the colonisation of indwelling medical devices by biofilm-forming opportunistic pathogens such as Candida albicans and Staphylococcus aureus. These organisms frequently co-infect, resulting in synergistic interactions with enhanced virulence and resistance to treatment. C. albicans and S. aureus readily form dual-species biofilms on silicone elastomers, a commonly used medical device material, yet the colonisation phenotypes of these organisms on such surfaces remains poorly understood. We developed a simple, optically tractable model to mimic the colonisation of indwelling medical devices to investigate C. albicans and S. aureus biofilm formation. The system utilises discs of a silicone elastomer embedded in agar, reflecting device-associated conditions and enabling high-resolution imaging of biofilms formed by C. albicans and S. aureus co-culture. Initial results using the silicone elastomer colonisation model reveal robust biofilm formation. These biofilms exhibited morphological differences between dual species biofilms formed by S. aureus co-cultures with either yeast- or hyphal-form C. albicans, indicating the impact of differing C. albicans cell morphotypes in biofilm-associated medical device colonization on silicone elastomers. Quantification of biofilm formation by crystal violet staining provided further validation of the system. These findings underscore the importance of developing tools for biofilm study which more closely resemble the infectious microenvironment, with our work detailing such a system which can be employed in further study to improve strategies against device-related HAIs.

Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study we investigate the structural and mechanical impacts of Ca2+ supplementation in alginate-dominated biofilms grown in vitro and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase and/or DNAse I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+ supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in non-trivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/563605v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@126dc6aorg.highwire.dtl.DTLVardef@50dab8org.highwire.dtl.DTLVardef@477fd6org.highwire.dtl.DTLVardef@19e8e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Antimicrobial resistance is a growing health problem. Pseudomonas aeruginosa is a pathogen of major concern because of its multidrug resistance and global threat, especially in health-care settings. The pathogenesis and drug resistance of P. aeruginosa depends on its ability to form biofilms, making infections chronic and untreatable as the biofilm protects against antibiotics and host immunity. A major barrier to developing new antimicrobials is the lack of in vivo biofilm models. Standard microbiological testing is usually performed in vitro using planktonic bacteria, without representation of biofilms, reducing translatability. Here we develop tools to study both infection and biofilm formation by P. aeruginosa in vivo to accelerate development of strategies targeting infection and pathogenic biofilms. Using the nematode Caenorhabditis elegans and P. aeruginosa reporters combined with in vivo imaging we show that fluorescent P. aeruginosa reporters that form biofilms in vitro can be used to visualise tissue infection. Using automated tracking of C. elegans movement, we find that that the timing of this infection corresponds with a decline in health endpoints. In a mutant strain of P. aeruginosa lacking RhlR, a transcription factor that controls quorum sensing and biofilm formation, we find reduced capacity of P. aeruginosa to form biofilms, invade host tissues and negatively impact healthspan and survival. Our findings suggest that RhlR could be a new antimicrobial target to reduce P. aeruginosa biofilms and virulence in vivo and C. elegans could be used to more effectively screen for new drugs to combat antimicrobial resistance.