{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T18:21:01Z","timestamp":1771611661338,"version":"3.50.1"},"reference-count":43,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,9,10]],"date-time":"2025-09-10T00:00:00Z","timestamp":1757462400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>Bacterial cell aggregation plays a fundamental role in surface colonization, stress tolerance, and interspecies metabolite exchange. Aggregation is assessed by simple tube-settling assays and also image analysis; however, approaches for quantitatively assessing the heterotypic and homotypic cell\u2013cell interactions among more than two types of cells have been limited. In this study, we developed grid partitioning image analysis (GPIA), a simple workflow that quantifies the compositional heterogeneity of bacterial aggregates. Confocal laser scanning microscopy (CLSM) images of fluorescently labeled <jats:italic>Acinetobacter<\/jats:italic> sp. Tol 5, which exhibits a self-aggregative nature through its cell surface protein AtaA, were partitioned into 2-\u03bcm square grids. Grids containing one or no cells were classified as dispersed, whereas those containing multiple cells were classified as aggregates, and the proportion of EGFP-labeled cells within each grid was recorded. Reference images representing dispersed cells, homo-aggregates, and hetero-aggregates produced characteristic EGFP-ratio histograms that matched binomial predictions. When AtaA production in one cell type was decreased, the histogram changed from a symmetric unimodal histogram with the peak at 40\u201360% EGFP-ratio to a skewed distribution, indicating that GPIA can detect differences in cell-to-cell affinity. Using the same procedure, we examined six in-frame deletion variants of AtaA. The deletion of the N-terminal head domain alone prevented co-aggregation with full-length AtaA, suggesting that homophilic recognition by this domain mediated self-aggregation, whereas deletions in all other regions had no measurable effect. GPIA, therefore, offers a simple and rapid approach for quantitative studies on bacterial cell aggregation, bridging the gap between qualitative microscopy and quantitative but technically demanding single-cell analysis. GPIA will accelerate research on cell\u2013cell interactions, which are the foundational processes that drive biofilm formation and the assembly of microbial consortia.<\/jats:p>","DOI":"10.3389\/fmicb.2025.1637462","type":"journal-article","created":{"date-parts":[[2025,9,10]],"date-time":"2025-09-10T18:15:22Z","timestamp":1757528122000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Grid partitioning image analysis of highly aggregative bacterium Acinetobacter sp. Tol 5"],"prefix":"10.3389","volume":"16","author":[{"given":"Yuki","family":"Ohara","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shogo","family":"Yoshimoto","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katsutoshi","family":"Hori","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,9,10]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"aaf5573","DOI":"10.1126\/science.aaf5573","article-title":"C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector","volume":"353","author":"Abudayyeh","year":"2016","journal-title":"Science"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"1195803","DOI":"10.3389\/fcimb.2023.1195803","article-title":"Imaging biofilms using fluorescence in situ hybridization: seeing is believing","volume":"13","author":"Barbosa","year":"2023","journal-title":"Front Cell Infect Micobiol"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"265","DOI":"10.1016\/j.ijmm.2014.12.010","article-title":"A domain dictionary of trimeric autotransporter adhesins","volume":"305","author":"Bassler","year":"2015","journal-title":"Int. J. Med. Microbiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"3950","DOI":"10.1128\/JB.06769-11","article-title":"Identification of Ata, a multifunctional trimeric autotransporter of Acinetobacter baumannii","volume":"194","author":"Bentancor","year":"2012","journal-title":"J. Bacteriol."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"2844","DOI":"10.1099\/ijs.0.027474-0","article-title":"Aquisphaera giovannonii gen. Nov., sp nov., a planctomycete isolated from a freshwater aquarium","volume":"61","author":"Bondoso","year":"2011","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"5887","DOI":"10.1007\/s11356-013-1824-5","article-title":"Quantitative image analysis for the characterization of microbial aggregates in biological wastewater treatment: a review","volume":"20","author":"Costa","year":"2013","journal-title":"Environ. Sci. Pollut. R."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"3815","DOI":"10.1038\/s41467-019-11725-5","article-title":"Cell morphology and nucleoid dynamics in dividing Deinococcus radiodurans","volume":"10","author":"Floc'h","year":"2019","journal-title":"Nat. Commun."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"3443","DOI":"10.1021\/acsnano.5b07515","article-title":"Sticky matrix: adhesion mechanism of the staphylococcal polysaccharide intercellular adhesin","volume":"10","author":"Formosa-Dague","year":"2016","journal-title":"ACS Nano"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"2520","DOI":"10.1021\/acs.est.9b06577","article-title":"Process description of an unconventional biofilm formation by bacterial cells autoagglutinating through sticky, long, and peritrichate nanofibers","volume":"54","author":"Furuichi","year":"2020","journal-title":"Environ. Sci. Technol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"649","DOI":"10.1016\/j.cell.2018.06.041","article-title":"A synthetic bacterial cell-cell adhesion toolbox for programming multicellular morphologies and patterns","volume":"174","author":"Glass","year":"2018","journal-title":"Cell"},{"key":"ref11","doi-asserted-by":"publisher","first-page":"1192","DOI":"10.1038\/s41592-021-01262-9","article-title":"DeepImageJ: a user-friendly environment to run deep learning models in ImageJ","volume":"18","author":"G\u00f3mez-de-Mariscal","year":"2021","journal-title":"Nat. Methods"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"e70018","DOI":"10.1002\/wer.70018","article-title":"Microalgal-bacterial aggregates for wastewater treatment: origins, challenges, and future directions","volume":"97","author":"Hammond","year":"2025","journal-title":"Water Environ. Res."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1038\/s41564-020-00817-4","article-title":"Quantitative image analysis of microbial communities with BiofilmQ","volume":"6","author":"Hartmann","year":"2021","journal-title":"Nat. Microbiol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"5989","DOI":"10.1093\/emboj\/19.22.5989","article-title":"Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins","volume":"19","author":"Hoiczyk","year":"2000","journal-title":"EMBO J."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"e00325","DOI":"10.1128\/jb.00325-24","article-title":"A new target of multiple lysine methylation in bacteria","volume":"207","author":"Inoue","year":"2025","journal-title":"J. Bacteriol."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"e48830","DOI":"10.1371\/journal.pone.0048830","article-title":"AtaA, a new member of the trimeric autotransporter adhesins from Acinetobacter sp. Tol 5 mediating high adhesiveness to various abiotic surfaces","volume":"7","author":"Ishikawa","year":"2012","journal-title":"PLoS One"},{"key":"ref17","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1002\/bit.25012","article-title":"Application of the adhesive bacterionanofiber AtaA to a novel microbial immobilization method for the production of indigo as a model chemical","volume":"111","author":"Ishikawa","year":"2014","journal-title":"Biotechnol. Bioeng."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1093\/femsre\/fuaa062","article-title":"Advances and opportunities in image analysis of bacterial cells and communities","volume":"45","author":"Jeckel","year":"2021","journal-title":"FEMS Microbiol. Rev."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"198","DOI":"10.1111\/j.1462-5822.2011.01711.x","article-title":"Analysis of the BadA stalk from Bartonella henselae reveals domain-specific and domain-overlapping functions in the host cell infection process","volume":"14","author":"Kaiser","year":"2012","journal-title":"Cell. Microbiol."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"e1109","DOI":"10.1002\/mbo3.1109","article-title":"Coaggregation properties of trimeric autotransporter adhesins","volume":"9","author":"Khalil","year":"2020","journal-title":"Microbiology"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"898","DOI":"10.1038\/s42003-023-05281-4","article-title":"The non-attached biofilm aggregate","volume":"6","author":"Kragh","year":"2023","journal-title":"Commun. Biol."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"1794","DOI":"10.1038\/s41396-020-00887-6","article-title":"Interspecies metabolite transfer and aggregate formation in a co-culture of Dehalococcoides and Sulfurospirillum dehalogenating tetrachloroethene to ethene","volume":"15","author":"Kruse","year":"2021","journal-title":"ISME J."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"1088","DOI":"10.1098\/rstb.2011.0208","article-title":"Type V secretion: mechanism(s) of autotransport through the bacterial outer membrane","volume":"367","author":"Leo","year":"2012","journal-title":"Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"2787","DOI":"10.1038\/s41467-025-58106-9","article-title":"Emergence and global spread of a dominant multidrug-resistant clade within Acinetobacter baumannii","volume":"16","author":"Li","year":"2025","journal-title":"Nat. Commun."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1038\/s44259-024-00046-3","article-title":"Mechanisms of antimicrobial resistance in biofilms","volume":"2","author":"Liu","year":"2024","journal-title":"NPJ Antimicrob Resist"},{"key":"ref26","doi-asserted-by":"publisher","first-page":"1203","DOI":"10.1093\/nar\/25.6.1203","article-title":"Independent and tight regulation of transcriptional units in Escherichia coli via the LacR\/O, the TetR\/O and AraC\/I-1-I-2 regulatory elements","volume":"25","author":"Lutz","year":"1997","journal-title":"Nucleic Acids Res."},{"key":"ref27","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1007\/978-94-007-0940-9_9","article-title":"Structure and biology of trimeric autotransporter adhesins","volume-title":"Bacterial adhesion","author":"Lyskowski","year":"2011"},{"key":"ref28","doi-asserted-by":"publisher","first-page":"e2021337","DOI":"10.4322\/acr.2021.337","article-title":"Sarcina ventriculi a rare pathogen","volume":"11","author":"Marcelino","year":"2021","journal-title":"Autops. Case Rep."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"1163","DOI":"10.3389\/fmicb.2019.01163","article-title":"Type V secretion systems: an overview of passenger domain functions","volume":"10","author":"Meuskens","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"1707","DOI":"10.1002\/jctb.7638","article-title":"Immobilization: the promising technique to protect and increase the efficiency of microorganisms to remove contaminants","volume":"99","author":"Najim","year":"2024","journal-title":"J. Chem. Technol. Biotechnol."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"1147","DOI":"10.1042\/BST20200718","article-title":"Bacteria autoaggregation: how and why bacteria stick together","volume":"49","author":"Nwoko","year":"2021","journal-title":"Biochem. Soc. Trans."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"1354","DOI":"10.1038\/s41467-020-15165-4","article-title":"Dynamics of bacterial population growth in biofilms resemble spatial and structural aspects of urbanization","volume":"11","author":"Paula","year":"2020","journal-title":"Nat. Commun."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1093\/femsre\/fuy040","article-title":"Fluorescence in situ hybridization, a complementary molecular tool for the clinical diagnosis of infectious diseases by intracellular and fastidious bacteria","volume":"43","author":"Prudent","year":"2019","journal-title":"FEMS Microbiol. Rev."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"628879","DOI":"10.3389\/fmicb.2021.628879","article-title":"BamA and BamD are essential for the secretion of trimeric autotransporter adhesins","volume":"12","author":"Rooke","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH image to ImageJ: 25 years of image analysis","volume":"9","author":"Schneider","year":"2012","journal-title":"Nat. Methods"},{"key":"ref36","doi-asserted-by":"publisher","first-page":"162213","DOI":"10.1016\/j.scitotenv.2023.162213","article-title":"Celebrating 50 years of microbial granulation technologies: from canonical wastewater management to bio-product recovery","volume":"872","author":"Sethi","year":"2023","journal-title":"Sci. Total Environ."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"e00620","DOI":"10.1128\/AEM.00620-19","article-title":"Fluorescence tools adapted for real-time monitoring of the behaviors of Streptococcus species","volume":"85","author":"Shields","year":"2019","journal-title":"Appl Environ Microb"},{"key":"ref38","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1083\/jcb.90.1.55","article-title":"Quantitative analysis of intercellular adhesive specificity in freshly explanted and cultured cells","volume":"90","author":"Sieber","year":"1981","journal-title":"J. Cell Biol."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"784","DOI":"10.1038\/nbt1183-784","article-title":"A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria","volume":"1","author":"Simon","year":"1983","journal-title":"Nat. Biotechnol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"2352","DOI":"10.1021\/acsinfecdis.1c00021","article-title":"The Acinetobacter baumannii autotransporter adhesin Ata recognizes host glycans as high-affinity receptors","volume":"7","author":"Tram","year":"2021","journal-title":"Acs Infect Dis"},{"key":"ref41","doi-asserted-by":"publisher","first-page":"140","DOI":"10.3934\/microbiol.2018.1.140","article-title":"Bacterial autoaggregation","volume":"4","author":"Trunk","year":"2018","journal-title":"AIMS Microbiol."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1080\/21505594.2018.1558693","article-title":"The Acinetobacter trimeric autotransporter adhesin Ata controls key virulence traits of Acinetobacter baumannii","volume":"10","author":"Weidensdorfer","year":"2019","journal-title":"Virulence"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"19557","DOI":"10.3389\/fbioe.2022.1095057","article-title":"Identification of the adhesive domain of AtaA from Acinetobacter sp. Tol 5 and its application in immobilizing Escherichia coli","volume":"10","author":"Yoshimoto","year":"2023","journal-title":"Front. Bioeng. Biotechnol."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1637462\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,9,10]],"date-time":"2025-09-10T18:15:24Z","timestamp":1757528124000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1637462\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,10]]},"references-count":43,"alternative-id":["10.3389\/fmicb.2025.1637462"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2025.1637462","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,10]]},"article-number":"1637462"}}