Extracellular host DNA contributes to pathogenic biofilm formation during periodontitis.

Introduction: Periodontal diseases are known to be associated with polymicrobial biofilms and inflammasome activation. A deeper understanding of the subgingival cytological (micro) landscape, the role of extracellular DNA (eDNA) during periodontitis, and contribution of the host immune eDNA to inflammasome persistence, may improve our understanding of the mechanisms underlaying severe forms of periodontitis. Methods: In this work, subgingival biolfilms developing on biologically neutral polyethylene terephthalate films placed in gingival cavities of patients with chronic periodontitis were investigated by confocal laser scanning microscopy (CLSM). This allowed examination of realistic cytological landscapes and visualization of extracellular polymeric substances (EPS) including amyloids, total proteins, carbohydrates and eDNA, as well as comparison with several single-strain in vitro model biofilms produced by oral pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus gordonii, S. sanguinis and S. mitis. Fluorescence in situ hybridization (FISH) analysis was also used to identify eDNA derived from eubacteria, streptococci and members of the Bacteroides-Porphyromonas-Prevotella (BPP) group associated with periodontitis. Results: Analysis of subgingival biofilm EPS revealed low levels of amyloids and high levels of eDNA which appears to be the main matrix component. However, bacterial eDNA contributed less than a third of the total eDNA observed, suggesting that host-derived eDNA released in neutrophil extracellular traps may be of more importance in the development of biofilms causing periodontitis. Discussion: eDNA derived from host immunocompetent cells activated at the onset of periodontitis may therefore be a major driver of bacterial persistence and pathogenesis.

Bioinformatics characterization of BcsA-like orphan proteins suggest they form a novel family of pseudomonad cyclic-ß-glucan synthases.

Bacteria produce a variety of polysaccharides with functional roles in cell surface coating, surface and host interactions, and biofilms. We have identified an 'Orphan' bacterial cellulose synthase catalytic subunit (BcsA)-like protein found in four model pseudomonads, P. aeruginosa PA01, P. fluorescens SBW25, P. putida KT2440 and P. syringae pv. tomato DC3000. Pairwise alignments indicated that the Orphan and BcsA proteins shared less than 41% sequence identity suggesting they may not have the same structural folds or function. We identified 112 Orphans among soil and plant-associated pseudomonads as well as in phytopathogenic and human opportunistic pathogenic strains. The wide distribution of these highly conserved proteins suggest they form a novel family of synthases producing a different polysaccharide. In silico analysis, including sequence comparisons, secondary structure and topology predictions, and protein structural modelling, revealed a two-domain transmembrane ovoid-like structure for the Orphan protein with a periplasmic glycosyl hydrolase family GH17 domain linked via a transmembrane region to a cytoplasmic glycosyltransferase family GT2 domain. We suggest the GT2 domain synthesises ß-(1,3)-glucan that is transferred to the GH17 domain where it is cleaved and cyclised to produce cyclic-ß-(1,3)-glucan (CßG). Our structural models are consistent with enzymatic characterisation and recent molecular simulations of the PaPA01 and PpKT2440 GH17 domains. It also provides a functional explanation linking PaPAK and PaPA14 Orphan (also known as NdvB) transposon mutants with CßG production and biofilm-associated antibiotic resistance. Importantly, cyclic glucans are also involved in osmoregulation, plant infection and induced systemic suppression, and our findings suggest this novel family of CßG synthases may provide similar range of adaptive responses for pseudomonads.

Investigating quorum-quenching marine bacilli as potential biocontrol agents for protection of shrimps against Early Mortality Syndrome (EMS).

Early Mortality Syndrome (EMS) has been a major problem for shrimp aquaculture in Southeast Asia due to its epizootic prevalence within the region since the first reported case in 2009. This study explores the application of halophilic marine bacilli isolated from coral mucus and their quorum-quenching abilities as potential biocontrol agents in aquaculture systems to combat the causative agent of EMS, Vibrio parahaemolyticus. N-acylhomoserine lactone (AHL)-degrading (AiiA) activity was first screened by PCR then confirmed by bio-reporter assay, and a combination of 16S rDNA sequence analysis and quantitative phenotype assays including biofilm-formation and temperature-growth responses were used to demonstrate diversity amongst these quorum-quenching isolates. Three phenotypically distinct strains showing notable potential were chosen to undergo co-cultivation as a method for strain improvement via long term exposure to the pathogenic V. parahaemolyticus. The novel approach taken led to significant improvements in antagonism and quorum quenching activities as compared to the ancestral wild-type strains and offers a potential solution as well as pathway to improve existing beneficial microbes for one of the most pressing issues in shrimp aquacultures worldwide.

Validation of a Salivary miRNA Signature of Endometriosis - Interim Data.

BACKGROUND: The discovery of a saliva-based micro­ribonucleic acid (miRNA) signature for endometriosis in 2022 opened up new perspectives for early and noninvasive diagnosis of the disease. The 109-miRNA saliva signature is the product of miRNA biomarkers and artificial intelligence (AI) modeling. We designed a multicenter study to provide external validation of its diagnostic accuracy. We present here an interim analysis. METHODS: The first 200 patients included in the multicenter prospective ENDOmiRNA Saliva Test study (NCT05244668) were included for interim analysis. The study population comprised women from 18 to 43 years of age with a formal diagnosis of endometriosis or with suspected endometriosis. Epidemiologic, clinical, and saliva sequencing data were collected between November 2021 and March 2022. Genomewide miRNA expression profiling by small RNA sequencing using next-generation sequencing (NGS) was performed, and a random forest algorithm was used to assess the diagnostic accuracy. RESULTS: In this interim analysis of the external validation cohort, with a population prevalence of 79.5%, the 109-miRNA saliva diagnostic signature for endometriosis had a sensitivity of 96.2% (95% confidence interval [CI], 93.7 to 97.3%), specificity of 95.1% (95% CI, 85.2 to 99.1%), positive predictive value of 95.1% (95% CI, 85.2 to 99.1%), negative predictive value of 86.7% (95% CI, 77.6 to 90.3%), positive likelihood ratio of 19.7 (95% CI, 6.3 to 108.8), negative likelihood ratio of 0.04 (95% CI, 0.03 to 0.07), and area under the receiver operating characteristic curve of 0.96 (95% CI, 0.92 to 0.98). CONCLUSIONS: The use of NGS and AI in the sequencing and analysis of miRNA provided a saliva-based miRNA signature for endometriosis. Our interim analysis of a prospective multicenter external validation study provides support for its ongoing investigation as a diagnostic tool. (Funded by Ziwig and the Conseil Régional d'Ile de France [Grant EX024087]; ClinicalTrials.gov number, NCT05244668.)

eDNA, Amyloid Fibers and Membrane Vesicles Identified in Pseudomonas fluorescens SBW25 Biofilms.

Pseudomonas fluorescens SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air-liquid interface biofilms in experimental microcosms and on plant surfaces. Previous investigations have shown that cellulose is the primary structural matrix component in the robust and well-attached Wrinkly Spreader biofilm, as well as in the fragile Viscous Mass biofilm. Here, we demonstrate that both biofilms include extracellular DNA (eDNA) which can be visualized using confocal laser scanning microscopy (CLSM), quantified by absorbance measurements, and degraded by DNase I treatment. This eDNA plays an important role in cell attachment and biofilm development. However, exogenous high-molecular-weight DNA appears to decrease the strength and attachment levels of mature Wrinkly Spreader biofilms, whereas low-molecular-weight DNA appears to have little effect. Further investigation with CLSM using an amyloid-specific fluorophore suggests that the Wrinkly Spreader biofilm might also include Fap fibers, which might be involved in attachment and contribute to biofilm strength. The robust nature of the Wrinkly Spreader biofilm also allowed us, using MALDI-TOF mass spectrometry, to identify matrix-associated proteins unable to diffuse out of the structure, as well as membrane vesicles which had a different protein profile compared to the matrix-associated proteins. CLSM and DNase I treatment suggest that some vesicles were also associated with eDNA. These findings add to our understanding of the matrix components in this model pseudomonad, and, as found in other biofilms, biofilm-specific products and material from lysed cells contribute to these structures through a range of complex interactions.

Azithromycin possesses biofilm-inhibitory activity and potentiates non-bactericidal colistin methanesulfonate (CMS) and polymyxin B against Klebsiella pneumonia.

Novel antibiotic combinations may act synergistically to inhibit the growth of multidrug-resistant bacterial pathogens but predicting which combination will be successful is difficult, and standard antimicrobial susceptibility testing may not identify important physiological differences between planktonic free-swimming and biofilm-protected surface-attached sessile cells. Using a nominally macrolide-resistant model Klebsiella pneumoniae strain (ATCC 10031) we demonstrate the effectiveness of several macrolides in inhibiting biofilm growth in multi-well plates, and the ability of azithromycin (AZM) to improve the effectiveness of the antibacterial last-agent-of-choice for K. pneumoniae infections, colistin methanesulfonate (CMS), against biofilms. This synergistic action was also seen in biofilm tests of several K. pneumoniae hospital isolates and could also be identified in polymyxin B disc-diffusion assays on azithromycin plates. Our work highlights the complexity of antimicrobial-resistance in bacterial pathogens and the need to test antibiotics with biofilm models where potential synergies might provide new therapeutic opportunities not seen in liquid culture or colony-based assays.

Late fetal demise, a risk factor for post-traumatic stress disorder.

Late-term fetal demise including fetal death in utero, late miscarriage and late termination of pregnancy are relatively frequent occurrences. Post-traumatic stress disorder (PTSD) is a pathology that finds its roots in exposure to a life-threatening event or an event related to death. Exposure to fetal death during a late-term fetal demise is, therefore, a situation at risk of trauma. The objective of this study was to assess the prevalence of PTSD symptoms in the short term among patients faced with late fetal demise, and to identify potential risk factors. All women were assessed at 15 days, one month, and three months after late fetal demise using the Impact of Event Scale-Revised (IES-R) and the Peritraumatic Dissociative Experiences Questionnaire (PDEQ). At 15 days, 44.2% of patients presented a pathological score on the IES-R (≥ 33). At one month and three months, this figure was 34.1% and 9.1% respectively. Factor associated with PTSD symptoms were: peritraumatic dissociation (p = 0.014), seeing the fetus during hospitalization (p = 0.035), holding the fetus in one's arms (p = 0.046), and the organization of a funeral service (p = 0.025). Patients experiencing late fetal demise are at significant risk of trauma. Care providers should remain vigilant to identify high-risk situations to offer appropriate care.Clinical trials registration number: NCT03433989.

eDNA Inactivation and Biofilm Inhibition by the PolymericBiocide Polyhexamethylene Guanidine Hydrochloride (PHMG-Cl).

The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA-PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.

Population level physical activity before and during the first national COVID-19 lockdown: A nationally representative repeat cross-sectional study of 5 years of Active Lives data in England.

BACKGROUND: To limit the spread of COVID-19 in March 2020, the population of England was instructed to stay home, leaving only for essential shopping, health-care, work, or exercise. The impact on population activity behaviours is not clear. We describe changes in duration and types of activity undertaken by adults ≥16 years in England between March and May 2016-19 and 2020, by socio-demographic strata. METHODS: Using nationally representative data collected between November 2015 and May 2020 by the Sport England Active Lives Surveys (n=726,257) we assessed trends in amount and type of non-occupational moderate-to-vigorous physical activity. Using data from n=74,430 mid-April to mid-May respondents, we then estimated the odds ratios of reporting any activity in the four-week recall period in 2020 compared to 2016-19. Gamma regressions estimated the mean ratios (MR) of duration amongst those reporting any activity in 2020 compared to 2016-19. FINDINGS: Population activity declined substantially after the restrictions were introduced. Compared to 2016-19 levels, the odds of reporting any activity in 2020 were 30% lower (95% confidence interval (CI) 26-34%). The largest declines were amongst non-white ethnicities, the youngest and oldest age groups, and the unemployed; no socio-demographic subgroup had higher odds. Amongst those undertaking activity, weekly duration was similar in the two periods (MR 0.99, 95%CI (0.96-1.01%)). The odds of participating in walking for leisure and gardening were 11% (6-16%) and 15% (9-21%) higher, respectively, whereas the odds for team and racket sport and walking for travel participation were 76% (73-79%) and 66% (64-68%) lower, respectively. INTERPRETATION: Restrictions introduced in Spring 2020 likely reduced physical activity levels in England. The magnitude of the declines were not uniform by demographic groups or by activity type, which future policies should consider. FUNDING: TS, KW, SJS, and SB are supported by UK Medical Research Council [grant numbers MC_UU_00006/4 and MC_UU_12015/3] and SB is supported by the NIHR Biomedical Research Centre in Cambridge (IS-BRC-1215-20014).

Efficacy of In Situ Methotrexate Injection in the Treatment of Nontubal Ectopic Pregnancies: A Retrospective Multicenter Study.

STUDY OBJECTIVE: Evaluation of the efficacy of different injection sites of methotrexate in the treatment of nontubal ectopic pregnancies. DESIGN: Retrospective multicenter study. SETTING: Multicenter, including 3 teaching hospitals, an intercommunal hospital, and a clinic. PATIENTS: A total of 106 patients with nontubal ectopic pregnancies, including 59 interstitial, 39 cesarean scar, and 8 cervical or isthmic. INTERVENTIONS: Overall, 58 patients received methotrexate via intramuscular injection (IM group), 35 received methotrexate via in situ injection (IS group), and 13 received a combination of both in situ and intramuscular injections of methotrexate (IS + IM group). MEASUREMENTS AND MAIN RESULTS: The main end point of this study was measured via the primary success rate (defined as a negative ß-human chorionic gonadotropin level without recourse to any additional treatment) of treatment with methotrexate according to injection site. The primary success rate was 46.55% in the IM group, 60% in the IS group, and 61.54% in the IS + IM group, respectively. In the multivariate analysis, the primary success rate of treatment was significantly correlated to the in situ injection of methotrexate, either solely or in conjunction with an intramuscular injection of methotrexate administered the following day, (odds ratio = 2.7; 95% confidence interval, 1.03-7.14). CONCLUSION: Solely an intramuscular injection of methotrexate is a less efficient first-line treatment strategy for the conservative management of nontubular ectopic pregnancy. The use of an in situ injection of methotrexate should therefore be preferred.

Ex utero intrapartum technique (EXIT): Indications, procedure methods and materno-fetal complications - A literature review.

A congenital malformation of the head, neck or thorax can lead to upper airway compression with a risk of asphyxia or neonatal death. To secure and protect the upper airway, the Ex Utero Intrapartum Therapy (EXIT) procedure has been developed. The procedure allows delivery of the fetus via a hysterotomy while relying on the placenta as the organ of respiration for the fetus prior to clamping of the umbilical cord. A high level of expertise is necessary for successful completion of the EXIT procedure, which is not void of maternal and fetal risks. In this literature review, we present the indications, procedure methods and materno-fetal complications associated with the EXIT procedure.

Delivery Mode After Manual Rotation of Occiput Posterior Fetal Positions: A Randomized Controlled Trial.

OBJECTIVE: To evaluate whether manual rotation of fetuses in occiput posterior positions at full dilation increases the rate of spontaneous vaginal delivery. METHODS: In an open, single-center, randomized controlled trial, patients with a term, singleton gestation, epidural analgesia, and ultrasonogram-confirmed occiput posterior position at the start of the second stage of labor were randomized to either manual rotation or expectant management. Our primary endpoint was the rate of spontaneous vaginal delivery. Secondary endpoints were operative vaginal delivery, cesarean delivery, and maternal and neonatal morbidity. Analyses were based on an intention-to-treat method. A sample size of 107 patients per group (n=214) was planned to detect a 20% increase in the percent of patients with a spontaneous vaginal delivery (assuming 60% without manual rotation vs 80% with manual rotation) with 90% power and alpha of 0.05. RESULTS: Between February 2017 and January 2020, 236 patients were randomized to either manual rotation (n=117) or expectant management (n=119). The success rate of the manual rotation maneuver, defined by conversion to an anterior position as confirmed by ultrasonogram, was 68%. The rate of the primary endpoint did not differ between the groups (58.1% in manual rotation group vs 59.7% in expectant management group (risk difference -1.6; 95% CI -14.1 to 11.0). Manual rotation did not decrease the rate of operative vaginal delivery (29.9% in manual rotation group vs 33.6% in expectant management group (risk difference -3.7; 95% CI -16.6 to 8.2) nor the rate of cesarean delivery (12.0% in manual rotation group vs 6.7% in expectant management group (risk difference 5.3; 95% CI -2.2 to 12.6). Maternal and neonatal morbidity was also similar across the two groups. CONCLUSION: Manual rotation of occiput posterior positions at the start of second stage of labor does not increase the rate of vaginal delivery without instrumental assistance. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT03009435.

External cephalic version: Predictors for success.

OBJECTIVES: The objective of this study was to identify the factors predictive of the success of external cephalic version (ECV). METHODS: A single-centre retrospective observational study was performed in the maternity ward of the Angers University Hospital, France, between January 2010 and May 2020. The study included all patients (n = 613) for whom an ECV was performed for a breech or transverse foetus. The primary endpoint was measured by the success of the ECV, defined by the visualisation, using pelvic ultrasound, of the foetus in cephalic presentation immediately after the manoeuvre. Following to the ECV, the cohort was separated into two groups; ECV Success and ECV Failure. In order to determine the predictive factors of success, a logistic regression model was performed, including the parameters of: parity, foetal presentation during ECV, the side of the foetal back, placental location, type of operator and maternal age. The results are presented as (odds ratio [confidence intervals]; p-value). The significance threshold was defined by a p-value < 0.05. RESULTS: The ECV success rate was 21.4% (131/613). The factors predictive of the success of ECV were: transverse foetal presentation (2.7 [1.3-5.6]; p<0.01); a senior physician operator (1.6 [1.2-2]; p<0.01); multiparity (1.6 [1.2-2]; p<0.01); non anterior placental localization (1.4 [1.1-2]; p<0.01). A number of attempts greater than 3 were significantly associated with reduced chances of success (0.3 [0.2-0.4]; p<0.01). CONCLUSION: The study shows that transverse foetal presentation, a senior physician operator, multiparity, as well as a non-anterior placental location are factors predictive of the success of ECV. Knowledge of these factors can improve the information given to patients.

The impact of the COVID-19 coronavirus pandemic on the surgical management of gynecological cancers: Analysis of the multicenter database of the French SCGP and the FRANCOGYN group.

INTRODUCTION: The coronavirus SARS-CoV-2 (COVID-19) pandemic has put tremendous pressure on the French healthcare system. Almost all hospital departments have had to profoundly modify their activity to cope with the crisis. In this context, the surgical management of cancers has been a topic of debate as care strategies were tailored to avoid any delay in treatment that could be detrimental to patient wellbeing while being careful not to overload intensive care units. The primary objective of this study was to observe changes in the surgical management of pelvic cancers during the COVID-19 pandemic in France. MATERIAL AND METHODS: This study analyzed data from the prospective multi-center cohort study conducted by the French Society for Pelvic and Gynecological Surgery (SCGP) with methodological support from the French (FRANCOGYN) Group. All members of the SCGP received by e-mail a link allowing them to include patients who were scheduled to undergo gynecological carcinologic surgery between March 16th 2020 and May 11th 2020. Demographic data, the characteristics of cancers and the impact of the crisis in terms of changes to the usual recommended coarse of care were collected. RESULTS: A total of 181 patients with a median age 63 years were included in the cohort. In total, 31 patients had cervical cancer, 76 patients had endometrial cancer, 52 patients had ovarian or tubal cancer, 5 patients had a borderline tumor of the ovary, and 17 patients had vulvar cancer. During the study period, the care strategy was changed for 49 (27%) patients with postponed for 35 (19.3%) patients, and canceled for 7 (3.9%) patients. Surgical treatment was maintained for 139 (76.8%) patients. Management with neoadjuvant chemotherapy was offered to 19 (10,5%) patients and a change in surgical choice was made for 5 (2,8%) patients. In total, 8 (4,4%) patients tested positive for COVID-19. Data also shows a greater number of therapeutic changes in cases of ovarian cancer as well as a cancelation of a lumbo-aortic lymphadenectomy in one patient with cervical cancer. Hospital consultants estimated a direct detrimental impact of the COVID-19 pandemic for 39 patients, representing 22% of gynecological cancers. CONCLUSION: This study provided observational data of the impact of the COVID-19 health crisis on the surgical management of gynecological cancers.

Mitochondria: their role in spermatozoa and in male infertility.

BACKGROUND: The best-known role of spermatozoa is to fertilize the oocyte and to transmit the paternal genome to offspring. These highly specialized cells have a unique structure consisting of all the elements absolutely necessary to each stage of fertilization and to embryonic development. Mature spermatozoa are made up of a head with the nucleus, a neck, and a flagellum that allows motility and that contains a midpiece with a mitochondrial helix. Mitochondria are central to cellular energy production but they also have various other functions. Although mitochondria are recognized as essential to spermatozoa, their exact pathophysiological role and their functioning are complex. Available literature relative to mitochondria in spermatozoa is dense and contradictory in some cases. Furthermore, mitochondria are only indirectly involved in cytoplasmic heredity as their DNA, the paternal mitochondrial DNA, is not transmitted to descendants. OBJECTIVE AND RATIONAL: This review aims to summarize available literature on mitochondria in spermatozoa, and, in particular, that with respect to humans, with the perspective of better understanding the anomalies that could be implicated in male infertility. SEARCH METHODS: PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews pertaining to human spermatozoa and mitochondria. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA', 'spermatozoa' or 'sperm' and 'reactive oxygen species' or 'calcium' or 'apoptosis' or signaling pathways'. These keywords were combined with other relevant search phrases. References from these articles were used to obtain additional articles. OUTCOMES: Mitochondria are central to the metabolism of spermatozoa and they are implicated in energy production, redox equilibrium and calcium regulation, as well as apoptotic pathways, all of which are necessary for flagellar motility, capacitation, acrosome reaction and gametic fusion. In numerous cases, alterations in one of the aforementioned functions could be linked to a decline in sperm quality and/or infertility. The link between the mitochondrial genome and the quality of spermatozoa appears to be more complex. Although the quantity of mtDNA, and the existence of large-scale deletions therein, are inversely correlated to sperm quality, the effects of mutations seem to be heterogeneous and particularly related to their pathogenicity. WIDER IMPLICATIONS: The importance of the role of mitochondria in reproduction, and particularly in gamete quality, has recently emerged following numerous publications. Better understanding of male infertility is of great interest in the current context where a significant decline in sperm quality has been observed.

Community biofilm-formation, stratification and productivity in serially-transferred microcosms.

The establishment of O2 gradients in liquid columns by bacterial metabolic activity produces a spatially-structured environment. This produces a high-O2 region at the top that represents an un-occupied niche which could be colonised by biofilm-competent strains. We have used this to develop an experimental model system using soil-wash inocula and a serial-transfer approach to investigate changes in community-based biofilm-formation and productivity. This involved 10 transfers of mixed-community or biofilm-only samples over a total of 10-60 days incubation. In all final-transfer communities the ability to form biofilms was retained, though in longer incubations the build-up of toxic metabolites limited productivity. Measurements of microcosm productivity, biofilm-strength and attachment levels were used to assess community-aggregated traits which showed changes at both the community and individual-strain levels. Final-transfer communities were stratified with strains demonstrating a plastic phenotype when migrating between the high and low-O2 regions. The majority of community productivity came from the O2-depleted region rather than the top of the liquid column. This model system illustrates the complexity we expect to see in natural biofilm-forming communities. The connection between biofilms and the liquid column seen here has important implications for how these structures form and respond to selective pressure.

Three biofilm types produced by a model pseudomonad are differentiated by structural characteristics and fitness advantage.

Model bacterial biofilm systems suggest that bacteria produce one type of biofilm, which is then modified by environmental and physiological factors, although the diversification of developing populations might result in the appearance of adaptive mutants producing altered structures with improved fitness advantage. Here we compare the air-liquid (A-L) interface viscous mass (VM) biofilm produced by Pseudomonas fluorescens SBW25 and the wrinkly spreader (WS) and complementary biofilm-forming strain (CBFS) biofilm types produced by adaptive SBW25 mutants in order to better understand the link between these physical structures and the fitness advantage they provide in experimental microcosms. WS, CBFS and VM biofilms can be differentiated by strength, attachment levels and rheology, as well as by strain characteristics associated with biofilm formation. Competitive fitness assays demonstrate that they provide similar advantages under static growth conditions but respond differently to increasing levels of physical disturbance. Pairwise competitions between biofilms suggest that these strains must be competing for at least two growth-limiting resources at the A-L interface, most probably O2 and nutrients, although VM and CBFS cells located lower down in the liquid column might provide an additional fitness advantage through the colonization of a less competitive zone below the biofilm. Our comparison of different SBW25 biofilm types illustrates more generally how varied biofilm characteristics and fitness advantage could become among adaptive mutants arising from an ancestral biofilm-forming strain and raises the question of how significant these changes might be in a range of medical, biotechnological and industrial contexts where diversification and change may be problematic.

Penetrating the air-liquid interface is the key to colonization and wrinkly spreader fitness.

In radiating populations of Pseudomonas fluorescens SBW25, adaptive wrinkly spreader (WS) mutants are able to gain access to the air-liquid (A-L) interface of static liquid microcosms and achieve a significant competitive fitness advantage over other non-biofilm-forming competitors. Aerotaxis and flagella-based swimming allows SBW25 cells to move into the high-O2 region located at the top of the liquid column and maintain their position by countering the effects of random cell diffusion, convection and disturbance (i.e. physical displacement). However, wild-type cells showed significantly lower levels of enrichment in this region compared to the archetypal WS, indicating that WS cells employ an additional mechanism to transfer to the A-L interface where displacement is no longer an issue and a biofilm can develop at the top of the liquid column. Preliminary experiments suggest that this might be achieved through the expression of an as yet unidentified surface active agent that is weakly associated with WS cells and alters liquid surface tension, as determined by quantitative tensiometry. The effect of physical displacement on the colonization of the high-O2 region and A-L interface was reduced through the addition of agar or polyethylene glycol to increase liquid viscosity, and under these conditions the competitive fitness of the WS was significantly reduced. These observations suggest that the ability to transfer to the A-L interface from the high-O2 region and remain there without further expenditure of energy (through, for example, the deployment of flagella) is a key evolutionary innovation of the WS, as it allows subsequent biofilm development and significant population increase, thereby affording these adaptive mutants a competitive fitness advantage over non-biofilm-forming competitors located within the liquid column.

Priming winter wheat seeds with the bacterial quorum sensing signal N-hexanoyl-L-homoserine lactone (C6-HSL) shows potential to improve plant growth and seed yield.

Several model plants are known to respond to bacterial quorum sensing molecules with altered root growth and gene expression patterns and induced resistance to plant pathogens. These compounds may represent novel elicitors that could be applied as seed primers to enhance cereal crop resistance to pathogens and abiotic stress and to improve yields. We investigated whether the acyl-homoserine lactone N-hexanoyl-L-homoserine lactone (C6-HSL) impacted winter wheat (Triticum aestivum L.) seed germination, plant development and productivity, using two Ukrainian varieties, Volodarka and Yatran 60, in both in vitro experiments and field trials. In vitro germination experiments indicated that C6-HSL seed priming had a small but significant positive impact on germination levels (1.2x increase, p < 0.0001), coleoptile and radicle development (1.4x increase, p < 0.0001). Field trials over two growing seasons (2015-16 and 2016-17) also demonstrated significant improvements in biomass at the tillering stage (1.4x increase, p < 0.0001), and crop structure and productivity at maturity including grain yield (1.4-1.5x increase, p < 0.0007) and quality (1.3x increase in good grain, p < 0.0001). In some cases variety effects were observed (p ≤ 0.05) suggesting that the effect of C6-HSL seed priming might depend on plant genetics, and some benefits of priming were also evident in F1 plants grown from seeds collected the previous season (p ≤ 0.05). These field-scale findings suggest that bacterial acyl-homoserine lactones such as C6-HSL could be used to improve cereal crop growth and yield and reduce reliance on fungicides and fertilisers to combat pathogens and stress.