scholarly journals Enterococcus faecalisbacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence ofCandida albicans

2017 ◽  
Vol 114 (17) ◽  
pp. 4507-4512 ◽  
Author(s):  
Carrie E. Graham ◽  
Melissa R. Cruz ◽  
Danielle A. Garsin ◽  
Michael C. Lorenz
Keyword(s):  
Biofilm Formation ◽  
Oral Microbiome ◽  
Ofcandida Albicans ◽  
Infection Models ◽  
Solid Substrates ◽  
Epithelial Invasion ◽  
Hyphal Formation ◽  
Biofilm Development ◽  
Necessary And Sufficient

Enterococcus faecalis, a Gram-positive bacterium, andCandida albicans, a fungus, occupy overlapping niches as ubiquitous constituents of the gastrointestinal and oral microbiome. Both species also are among the most important and problematic, opportunistic nosocomial pathogens. Surprisingly, these two species antagonize each other’s virulence in both nematode infection and in vitro biofilm models. We report here the identification of theE. faecalisbacteriocin, EntV, produced from theentV(ef1097) locus, as both necessary and sufficient for the reduction ofC. albicansvirulence and biofilm formation through the inhibition of hyphal formation, a critical virulence trait. A synthetic version of the mature 68-aa peptide potently blocks biofilm development on solid substrates in multiple media conditions and disrupts preformed biofilms, which are resistant to current antifungal agents. EntV68is protective in three fungal infection models at nanomolar or lower concentrations. First, nematodes treated with the peptide at 0.1 nM are completely resistant to killing byC. albicans. The peptide also protects macrophages and augments their antifungal activity. Finally, EntV68reduces epithelial invasion, inflammation, and fungal burden in a murine model of oropharyngeal candidiasis. In all three models, the peptide greatly reduces the number of fungal cells present in the hyphal form. Despite these profound effects, EntV68has no effect onC. albicansviability, even in the presence of significant host-mimicking stresses. These findings demonstrate that EntV has potential as an antifungal agent that targets virulence rather than viability.

scholarly journals Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species

2010 ◽  
Vol 59 (10) ◽  
pp. 1225-1234 ◽  
Author(s):  
H. M. H. N. Bandara ◽  
O. L. T. Lam ◽  
R. M. Watt ◽  
L. J. Jin ◽  
L. P. Samaranayake
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Significant Inhibition ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Bacterial Lipopolysaccharides ◽  
Species Specific ◽  
Biofilm Development

The objective of this study was to evaluate the effect of the bacterial endotoxin LPS on Candida biofilm formation in vitro. The effect of the LPS of Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium on six different species of Candida, comprising Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA 646, was studied using a standard biofilm assay. The metabolic activity of in vitro Candida biofilms treated with LPS at 90 min, 24 h and 48 h was quantified by XTT reduction assay. Viable biofilm-forming cells were qualitatively analysed using confocal laser scanning microscopy (CLSM), while scanning electron microscopy (SEM) was employed to visualize the biofilm structure. Initially, adhesion of C. albicans was significantly stimulated by Pseudomonas and Klebsiella LPS. A significant inhibition of Candida adhesion was noted for the following combinations: C. glabrata with Pseudomonas LPS, C. tropicalis with Serratia LPS, and C. glabrata, C. parapsilosis or C. dubliniensis with Salmonella LPS (P<0.05). After 24 h of incubation, a significant stimulation of initial colonization was noted for the following combinations: C. albicans/C. glabrata with Klebsiella LPS, C. glabrata/C. tropicalis/C. krusei with Salmonella LPS. In contrast, a significant inhibition of biofilm formation was observed in C. glabrata/C. dubliniensis/C. krusei with Pseudomonas LPS, C. krusei with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. parapsilosis/C. dubliniensis /C. krusei with Salmonella LPS (P<0.05). On further incubation for 48 h, a significant enhancement of biofilm maturation was noted for the following combinations: C. glabrata/C. tropicalis with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. glabrata with Salmonella LPS, and a significant retardation was noted for C. parapsilosis/C. dubliniensis/C. krusei with Pseudomonas LPS, C. tropicalis with Serratia LPS, C. glabrata/C. parapsilosis/C. dubliniensis with Klebsiella LPS and C. dubliniensis with Salmonella LPS (P<0.05). These findings were confirmed by SEM and CLSM analyses. In general, the inhibition of the biofilm development of LPS-treated Candida spp. was accompanied by a scanty architecture with a reduced numbers of cells compared with the profuse and densely colonized control biofilms. These data are indicative that bacterial LPSs modulate in vitro Candida biofilm formation in a species-specific and time-dependent manner. The clinical and the biological relevance of these findings have yet to be explored.

scholarly journals Bacterial diversity and functional analysis of severe early childhood caries and recurrence in India

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Balakrishnan Kalpana ◽  
Puniethaa Prabhu ◽  
Ashaq Hussain Bhat ◽  
Arunsaikiran Senthilkumar ◽  
Raj Pranap Arun ◽  
...  
Keyword(s):  
Early Childhood ◽  
Bacterial Diversity ◽  
Biofilm Formation ◽  
Early Childhood Caries ◽  
Oral Microbiome ◽  
Oral Disease ◽  
Indian Children ◽  
Severe Early Childhood Caries ◽  
Childhood Caries

AbstractDental caries is the most prevalent oral disease affecting nearly 70% of children in India and elsewhere. Micro-ecological niche based acidification due to dysbiosis in oral microbiome are crucial for caries onset and progression. Here we report the tooth bacteriome diversity compared in Indian children with caries free (CF), severe early childhood caries (SC) and recurrent caries (RC). High quality V3–V4 amplicon sequencing revealed that SC exhibited high bacterial diversity with unique combination and interrelationship. Gracillibacteria_GN02 and TM7 were unique in CF and SC respectively, while Bacteroidetes, Fusobacteria were significantly high in RC. Interestingly, we found Streptococcus oralis subsp. tigurinus clade 071 in all groups with significant abundance in SC and RC. Positive correlation between low and high abundant bacteria as well as with TCS, PTS and ABC transporters were seen from co-occurrence network analysis. This could lead to persistence of SC niche resulting in RC. Comparative in vitro assessment of biofilm formation showed that the standard culture of S. oralis and its phylogenetically similar clinical isolates showed profound biofilm formation and augmented the growth and enhanced biofilm formation in S. mutans in both dual and multispecies cultures.

scholarly journals Effects of CO2laser irradiation on matrix-rich biofilm development formation–an in vitro study

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2458 ◽  
Author(s):  
Bruna Raquel Zancopé ◽  
Vanessa B. Dainezi ◽  
Marinês Nobre-dos-Santos ◽  
Sillas Duarte ◽  
Vanessa Pardi ◽  
...  
Keyword(s):  
Contact Angle ◽  
Biofilm Formation ◽  
Dental Enamel ◽  
In Vitro Study ◽  
Dry Weight ◽  
Vitro Study ◽  
Enamel Surface ◽  
Control Group ◽  
Biofilm Development

BackgroundA carbon dioxide (CO2) laser has been used to morphologically and chemically modify the dental enamel surface as well as to make it more resistant to demineralization. Despite a variety of experiments demonstrating the inhibitory effect of a CO2laser in reduce enamel demineralization, little is known about the effect of surface irradiated on bacterial growth. Thus, this in vitro study was preformed to evaluate the biofilm formation on enamel previously irradiated with a CO2laser (λ = 10.6 µM).MethodsFor this in vitro study, 96 specimens of bovine enamel were employed, which were divided into two groups (n = 48): 1) Control-non-irradiated surface and 2) Irradiated enamel surface. Biofilms were grown on the enamel specimens by one, three and five days under intermittent cariogenic condition in the irradiated and non-irradiated surface. In each assessment time, the biofilm were evaluated by dry weigh, counting the number of viable colonies and, in fifth day, were evaluated by polysaccharides analysis, quantitative real time Polymerase Chain Reaction (PCR) as well as by contact angle. In addition, the morphology of biofilms was characterized by fluorescence microscopy and field emission scanning electron microscopy (FESEM). Initially, the assumptions of equal variances and normal distribution of errors were conferred and the results are analyzed statistically by t-test and Mann Whitney test.ResultsThe mean of log CFU/mL obtained for the one-day biofilm evaluation showed that there is statistical difference between the experimental groups. When biofilms were exposed to the CO2laser, CFU/mL and CFU/dry weight in three day was reduced significantly compared with control group. The difference in the genes expression (Glucosyltransferases (gtfB) and Glucan-binding protein (gbpB)) and polysaccharides was not statically significant. Contact angle was increased relative to control when the surface was irradiated with the CO2laser. Similar morphology was also visible with both treatments; however, the irradiated group revealed evidence of melting and fusion in the specimens.ConclusionIn conclusion, CO2laser irradiation modifies the energy surface and disrupts the initial biofilm formation.

scholarly journals Role of the Streptococcus mutans irvA Gene in GbpC-Independent, Dextran-Dependent Aggregation and Biofilm Formation

2009 ◽  
Vol 75 (22) ◽  
pp. 7037-7043 ◽  
Author(s):  
Min Zhu ◽  
Dragana Ajdić ◽  
Yuan Liu ◽  
David Lynch ◽  
Justin Merritt ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Growth Conditions ◽  
Parental Background ◽  
A Cell ◽  
Major Surface ◽  
Biofilm Development ◽  
Small Aggregation

ABSTRACT Dextran-dependent aggregation (DDAG) of Streptococcus mutans is an in vitro phenomenon that is believed to represent a property of the organism that is beneficial for sucrose-dependent biofilm development. GbpC, a cell surface glucan-binding protein, is responsible for DDAG in S. mutans when cultured under defined stressful conditions. Recent reports have described a putative transcriptional regulator gene, irvA, located just upstream of gbpC, that is normally repressed by the product of an adjacent gene, irvR. When repression of irvA is relieved, there is a resulting increase in the expression of GbpC and decreases in competence and synthesis of the antibiotic mutacin I. This study examined the role of irvA in DDAG and biofilm formation by engineering strains that overexpressed irvA (IrvA+) on an extrachromosomal plasmid. The IrvA+ strain displayed large aggregation particles that did not require stressful growth conditions. A novel finding was that overexpression of irvA in a gbpC mutant background retained a measure of DDAG, albeit very small aggregation particles. Biofilms formed by the IrvA+ strain in the parental background possessed larger-than-normal microcolonies. In a gbpC mutant background, the overexpression of irvA reversed the fragile biofilm phenotype normally associated with loss of GbpC. Real-time PCR and Northern blot analyses found that expression of gbpC did not change significantly in the IrvA+ strain but expression of spaP, encoding the major surface adhesin P1, increased significantly. Inactivation of spaP eliminated the small-particle DDAG. The results suggest that IrvA promotes DDAG not only by GbpC, but also via an increase in P1.

scholarly journals Searching Hit Potential Antimicrobials in Natural Compounds Space against Biofilm Formation

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5334
Author(s):  
Roberto Pestana-Nobles ◽  
Jorge A. Leyva-Rojas ◽  
Juvenal Yosa
Keyword(s):  
Natural Products ◽  
Biofilm Formation ◽  
Caulobacter Crescentus ◽  
Resistant Bacteria ◽  
Chronic Infections ◽  
Aconitic Acid ◽  
Starting Point ◽  
In Silico Studies ◽  
Biofilm Development

Biofilms are communities of microorganisms that can colonize biotic and abiotic surfaces and thus play a significant role in the persistence of bacterial infection and resistance to antimicrobial. About 65% and 80% of microbial and chronic infections are associated with biofilm formation, respectively. The increase in infections by multi-resistant bacteria instigates the need for the discovery of novel natural-based drugs that act as inhibitory molecules. The inhibition of diguanylate cyclases (DGCs), the enzyme implicated in the synthesis of the second messenger, cyclic diguanylate (c-di-GMP), involved in the biofilm formation, represents a potential approach for preventing the biofilm development. It has been extensively studied using PleD protein as a model of DGC for in silico studies as virtual screening and as a model for in vitro studies in biofilms formation. This study aimed to search for natural products capable of inhibiting the Caulobacter crescentus enzyme PleD. For this purpose, 224,205 molecules from the natural products ZINC15 database, have been evaluated through molecular docking and molecular dynamic simulation. Our results suggest trans-Aconitic acid (TAA) as a possible starting point for hit-to-lead methodologies to obtain new inhibitors of the PleD protein and hence blocking the biofilm formation.

scholarly journals Early Biofilm Formation on UV Light Activated Nanoporous TiO2SurfacesIn Vivo

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Nagat Areid ◽  
Eva Söderling ◽  
Johanna Tanner ◽  
Ilkka Kangasniemi ◽  
Timo O. Närhi
Keyword(s):  
Titanium Alloy ◽  
Biofilm Formation ◽  
Uv Light ◽  
Antibacterial Properties ◽  
Mutans Streptococci ◽  
Human Gingival Fibroblast ◽  
Gingival Fibroblast ◽  
Biofilm Development

Purpose. To explore earlyS. mutansbiofilm formation on hydrothermally induced nanoporous TiO2surfacesin vivoand to examine the effect of UV light activation on the biofilm development.Materials and Methods. Ti-6Al-4V titanium alloy discs (n = 40) were divided into four groups with different surface treatments: noncoated titanium alloy (NC); UV treated noncoated titanium alloy (UVNC); hydrothermally induced TiO2coating (HT); and UV treated titanium alloy with hydrothermally induced TiO2coating (UVHT).In vivoplaque formation was studied in 10 healthy, nonsmoking adult volunteers. Titanium discs were randomly distributed among the maxillary first and second molars. UV treatment was administered for 60 min immediately before attaching the discs in subjects’ molars. Plaque samples were collected 24h after the attachment of the specimens. Mutans streptococci (MS), non-mutans streptococci, and total facultative bacteria were cultured, and colonies were counted.Results. The plaque samples of NC (NC + UVNC) surfaces showed over 2 times more oftenS. mutanswhen compared to TiO2surfaces (HT + UVHT), with the number of colonized surfaces equal to 7 and 3, respectively.Conclusion. Thisin vivostudy suggested that HT TiO2surfaces, which we earlier showed to improve blood coagulation and encourage human gingival fibroblast attachmentin vitro, do not enhance salivary microbial (mostly mutans streptococci) adhesion and initial biofilm formation when compared with noncoated titanium alloy. UV light treatment provided Ti-6Al-4V surfaces with antibacterial properties and showed a trend towards less biofilm formation when compared with non-UV treated titanium surfaces.

scholarly journals Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Jayajit Das ◽  
Elaine Mokrzan ◽  
Vinal Lakhani ◽  
Lucia Rosas ◽  
Joseph A. Jurcisek ◽  
...  
Keyword(s):  
Middle Ear ◽  
In Silico ◽  
Biofilm Formation ◽  
Rational Design ◽  
Bacterial Biofilm ◽  
Confocal Imaging ◽  
Extracellular Dna ◽  
Fractal Structures ◽  
Biofilm Development

ABSTRACT Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies.

Biofilm Formation by Candida Species on Silicone Surfaces and Latex Pacifier Nipples: An in vitro Study

2009 ◽  
Vol 33 (3) ◽  
pp. 235-240 ◽  
Author(s):  
Luiz Cezar da Silveira ◽  
Senda Charone ◽  
Lucianne Cople Maia ◽  
Rosangela Maria de Araújo Soares ◽  
Maristela Barbosa Portela
Keyword(s):  
Candida Species ◽  
Biofilm Formation ◽  
In Vitro Study ◽  
Reduction Reaction ◽  
Fungal Colonization ◽  
Candida Spp ◽  
Biofilm Growth ◽  
Species Analysis ◽  
Biofilm Development

The present study assessed the growth and development of biofilm formation by isolates of C. albicans, C. glabrata and C. parapsilosis on silicone and latex pacifier nipples. The silicone and latex surfaces were evaluated by scanning electronic microscopy (SEM). The plastic component of the nipple also seems to be an important factor regarding the biofilm formation by Candida spp. The biofilm growth was measured using the MTT reduction reaction. C. albicans was found to have a slightly greater capacity of forming biofilm compared to the other Candida species. Analysis of the pattern of biofilm development by C. albicans,C. glabrata and C. parapsilosis on latex and silicon pacifier shields showed an increased biofilm formation regarding the latter substrate. Silicone was shown to be more resistant to fungal colonization, particularly in the case of C. parapsilosis, despite the lack of any statistically significant differences (P &gt; 0.05). In addition, silicone has a smoother surface compared to latex, whose surface was found to be rugose and irregular

scholarly journals Modulation of antibiotic sensitivity and biofilm formation in Pseudomonas aeruginosa by interspecies diffusible signal factor analogues

2018 ◽  
Author(s):  
Shi-qi An ◽  
Julie Murtagh ◽  
Kate B. Twomey ◽  
Manoj K. Gupta ◽  
Timothy P. O’Sullivan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Unsaturated Fatty Acids ◽  
Antibiotic Sensitivity ◽  
Opportunistic Pathogen ◽  
Lead Compounds ◽  
Infection Models ◽  
Diffusible Signal Factor ◽  
Input Domain

ABSTRACTThe opportunistic pathogen Pseudomonas aeruginosa can participate in inter-species communication through signaling by cis-2-unsaturated fatty acids of the diffusible signal factor (DSF) family. Sensing these signals involves the histidine kinase PA1396 and leads to altered biofilm formation and increased tolerance to various antibiotics. Here, we show that the membrane-associated sensory input domain of PA1396 has five trans-membrane helices, two of which are required for DSF sensing. DSF binding is associated with enhanced auto-phosphorylation of PA1396 incorporated into liposomes. Further, we examined the ability of synthetic DSF analogues to modulate or inhibit PA1396 activity. Several of these analogues block the ability of DSF to trigger auto-phosphorylation and gene expression, whereas others act as inverse agonists reducing biofilm formation and antibiotic tolerance, both in vitro and in murine infection models. These analogues may thus represent lead compounds for novel adjuvants to improve the efficacy of existing antibiotics.

scholarly journals The cyclic di-GMP network is a global regulator of phase-transition and attachment-dependent host colonization in Erwinia amylovora

2021 ◽  
Author(s):  
Roshni R. Kharadi ◽  
Kayla Selbmann ◽  
George W. Sundin
Keyword(s):  
Signal Transduction ◽  
Biofilm Formation ◽  
Erwinia Amylovora ◽  
Initial Surface ◽  
Diguanylate Cyclase ◽  
Wide Range ◽  
Biofilm Development ◽  
Bacterial Systems

AbstractCyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates the transition to biofilm formation in the phytopathogen Erwinia amylovora. The c-di-GMP system in E. amylovora is comprised of 12 diguanylate cyclase/Edc (dimerize cyclic-di-GMP) and phosphodiesterase/Pde (hydrolyze cyclic-di-GMP) proteins that are characterized by the presence of GGDEF and/or EAL motifs in their domain architecture. In order to study the global regulatory effect (without the inclusion of systemic regulatory impedance) of the c-di-GMP system in E. amylovora, we eliminated all 12 edc and pde genes in E. amylovora Ea1189Δ12. Comparisons between the representative transcriptomic profiles of Ea1189Δ12 and the combinatorial edc gene knockout mutant (Ea1189Δ5) revealed marked overall distinctions in expression levels for targets in a wide range of regulatory categories, including metabolic pathways involved in the utilization of methionine, isoleucine, histidine, etc. as well as critical signal transduction pathways including the Rcs phosphorelay and PhoPQ system. A complete loss of the cyclic-di-GMP signaling components resulted in the inability of Ea1189Δ12 cells to attach to and form biofilms in vitro and within the xylem vasculature in apple shoots. Using a flow-based in vitro biofilm system, we found that initial surface sensing was primarily dependent on the flagellar filament (FliC), following which the type IV pilus (HofC) was required to anchor cells to the surface to initialize biofilm development. A transcriptomic analysis of WT E. amylovora Ea1189 and Ea1189Δ12 cells in various stages of biofilm development revealed that cyclic-di-GMP based regulation had widespread effects on purine and pyrimidine biosynthesis pathways, amylovoran biosynthesis genes and the EnvZ/OmpR signal transduction system. Additionally, complementing individual eliminated genes back into Ea1189Δ12, and the collective evaluation of several virulence factors, enabled the correlative clustering of the functional effect rendered by each Edc and Pde enzyme in the system.SignificanceCyclic-di-GMP dependent regulation, in the context of biofilm formation, has been studied in several bacterial systems. However, the comprehensiveness of the studies exploring the role of individual genetic components related to cyclic-di-GMP is affected by the often large number of diguanylate cyclase and phosphodiesterase enzymes present within individual bacterial systems. To explore the evolutionary dependencies related to cyclic-di-GMP in E. amylovora, we used a collective elimination approach, whereby all of the enzymes involved in cyclic-di-GMP metabolism were eliminated from the system. This approach enabled us to highlight the critical importance of cyclic-di-GMP in plant xylem colonization due to its effect on surface attachment. Additionally, we highlight the global transcriptomic effect of cyclic-di-GMP dependent signaling at various stages of biofilm development. Our approach is aimed at exploring the regulatory role of individual cyclic-di-GMP related enzymes in a background that is free from any redundancy-based feedback.

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