{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,18]],"date-time":"2026-01-18T22:44:18Z","timestamp":1768776258262,"version":"3.49.0"},"reference-count":46,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2024,8,9]],"date-time":"2024-08-09T00:00:00Z","timestamp":1723161600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Conahcyt","award":["992577"],"award-info":[{"award-number":["992577"]}]},{"name":"Conahcyt","award":["CB-A1-S-8989"],"award-info":[{"award-number":["CB-A1-S-8989"]}]},{"name":"Conahcyt","award":["992577"],"award-info":[{"award-number":["992577"]}]},{"name":"Conahcyt","award":["CB-A1-S-8989"],"award-info":[{"award-number":["CB-A1-S-8989"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Respiratory diseases in ruminants are responsible for enormous economic losses for the dairy and meat industry. The main causative bacterial agent of pneumonia in ovine is Mannheimia haemolytica A2. Due to the impact of this disease, the effect of the antimicrobial protein, bovine lactoferrin (bLf), against virulence factors of this bacterium has been studied. However, its effect on biofilm formation has not been reported. In this work, we evaluated the effect on different stages of the biofilm. Our results reveal a decrease in biofilm formation when bacteria were pre-incubated with bLf. However, when bLf was added at the start of biofilm formation and on mature biofilm, an increase was observed, which was visualized by greater bacterial aggregation and secretion of biofilm matrix components. Additionally, through SDS-PAGE, a remarkable band of ~80 kDa was observed when bLf was added to biofilms. Therefore, the presence of bLf on the biofilm was determined through the Western blot and Microscopy techniques. Finally, by using Live\/Dead staining, we observed that most of the bacteria in a biofilm with bLf were not viable. In addition, bLf affects the formation of a new biofilm cycle. In conclusion, bLf binds to the biofilm of M. haemolytica A2 and affects the viability of bacteria and the formation a new biofilm cycle.<\/jats:p>","DOI":"10.3390\/ijms25168718","type":"journal-article","created":{"date-parts":[[2024,8,14]],"date-time":"2024-08-14T03:46:36Z","timestamp":1723607196000},"page":"8718","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Lactoferrin Affects the Viability of Bacteria in a Biofilm and the Formation of a New Biofilm Cycle of Mannheimia haemolytica A2"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7376-6558","authenticated-orcid":false,"given":"Lucero","family":"Ruiz-Maz\u00f3n","sequence":"first","affiliation":[{"name":"Departamento de Biolog\u00eda Celular, Centro de Investigaci\u00f3n y de Estudios Avanzados del Instituto Polit\u00e9cnico Nacional (CINVESTAV-IPN), Ciudad de Mexico 07360, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2546-2792","authenticated-orcid":false,"given":"Gerardo","family":"Ram\u00edrez-Rico","sequence":"additional","affiliation":[{"name":"Departamento de Biolog\u00eda Celular, Centro de Investigaci\u00f3n y de Estudios Avanzados del Instituto Polit\u00e9cnico Nacional (CINVESTAV-IPN), Ciudad de Mexico 07360, Mexico"},{"name":"Facultad de Estudios Superiores Cuautitl\u00e1n, Universidad Nacional Aut\u00f3noma de M\u00e9xico (UNAM), Km 2.5 Carretera Cuautitl\u00e1n-Teoloyucan, Cuautitl\u00e1n Izcalli 54714, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mireya","family":"de la Garza","sequence":"additional","affiliation":[{"name":"Departamento de Biolog\u00eda Celular, Centro de Investigaci\u00f3n y de Estudios Avanzados del Instituto Polit\u00e9cnico Nacional (CINVESTAV-IPN), Ciudad de Mexico 07360, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,8,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1016\/j.micpath.2017.09.061","article-title":"Bacterial, PCR and Clinico-Pathological Diagnosis of Naturally Occurring Pneumonic Pasturellosis (Mannheimiosis) during Subtropical Climate in Sheep","volume":"112","author":"Hussain","year":"2017","journal-title":"Microb. Pathog."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"5605552","DOI":"10.1155\/2024\/5605552","article-title":"Isolation and Identification of Pasteurella multocida and Mannheimia haemolytica from Pneumonic Small Ruminants and Their Antibiotic Susceptibility in Haramaya District, Eastern Ethiopia","volume":"2024","author":"Abdulkadir","year":"2024","journal-title":"Sci. World J."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1177\/0300985810377182","article-title":"Mannheimia haemolytica: Bacterial-Host Interactions in Bovine Pneumonia","volume":"48","author":"Singh","year":"2011","journal-title":"Vet. Pathol."},{"key":"ref_4","first-page":"1128","article-title":"Molecular Genetic Analysis of Virulence in Mannheimia (pasteurella) haemolytica","volume":"6","author":"Highlander","year":"2001","journal-title":"Front. Biosci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"105940","DOI":"10.1016\/j.tvjl.2022.105940","article-title":"Distinct Mannheimia haemolytica Serotypes Isolated from Fatal Infections in Veal Calves and Dairy Cows","volume":"292","author":"Derkman","year":"2023","journal-title":"Vet. J."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1446","DOI":"10.1128\/iai.61.11.4669-4674.1993","article-title":"Characterization of Neuraminidases Produced by Various Serotypes of Pasteurella haemolytica","volume":"61","author":"Straus","year":"1993","journal-title":"Infect. Immun."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1051\/vetres:2004065","article-title":"How Mannheimia haemolytica Defeats Host Defence through a Kiss of Death Mechanism","volume":"36","author":"Zecchinon","year":"2005","journal-title":"Vet. Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1016\/j.micpath.2017.10.027","article-title":"Mannheimia haemolytica A2 Secretes Different Proteases into the Culture Medium and in Outer Membrane Vesicles","volume":"113","year":"2017","journal-title":"Microb. Pathog."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Kragh, K.N., Tolker-Nielsen, T., and Lichtenberg, M. (2023). The Non-Attached Biofilm Aggregate. Commun. Biol., 6.","DOI":"10.1038\/s42003-023-05281-4"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Yin, W., Wang, Y., Liu, L., and He, J. (2019). Biofilms: The Microbial \u201cProtective Clothing\u201d in Extreme Environments. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20143423"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1743","DOI":"10.2217\/fmb.15.69","article-title":"Bacterial Resistance in Biofilm-Associated Bacteria","volume":"10","author":"Venkatesan","year":"2015","journal-title":"Future Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1016\/j.vetmic.2014.11.012","article-title":"Characterization of Mannheimia haemolytica Biofilm Formation in Vitro","volume":"175","author":"Boukahil","year":"2015","journal-title":"Vet. Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"522","DOI":"10.1080\/21505594.2017.1313372","article-title":"Strategies for Combating Bacterial Biofilms: A Focus on Anti-Biofilm Agents and Their Mechanisms of Action","volume":"9","author":"Roy","year":"2018","journal-title":"Virulence"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1007\/s12033-021-00325-8","article-title":"Novel Strategies to Combat Bacterial Biofilms","volume":"63","author":"Hemmati","year":"2021","journal-title":"Mol. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Abdelhamid, A.G., and Yousef, A.E. (2023). Combating Bacterial Biofilms: Current and Emerging Antibiofilm Strategies for Treating Persistent Infections. Antibiotics, 12.","DOI":"10.3390\/antibiotics12061005"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Grooters, K.E., Ku, J.C., Richter, D.M., Krinock, M.J., Minor, A., Li, P., Kim, A., Sawyer, R., and Li, Y. (2024). Strategies for Combating Antibiotic Resistance in Bacterial Biofilms. Front. Cell. Infect. Microbiol., 14.","DOI":"10.3389\/fcimb.2024.1352273"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1093\/femsre\/fux010","article-title":"Molecular Mechanisms of Biofilm-Based Antibiotic Resistance and Tolerance in Pathogenic Bacteria","volume":"41","author":"Hall","year":"2017","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3243","DOI":"10.2174\/1381612829666221025153216","article-title":"Lactoferrin: An Effective Weapon in the Battle Against Bacterial Infections","volume":"28","author":"Sicairos","year":"2022","journal-title":"Curr. Pharm. Des."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1548","DOI":"10.1128\/AAC.05100-11","article-title":"Lactoferrin Inhibits Porphyromonas gingivalis Proteinases and Has Sustained Biofilm Inhibitory Activity","volume":"56","author":"Dashper","year":"2012","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3308","DOI":"10.1128\/AAC.01688-08","article-title":"Inhibitory Effects of Lactoferrin on Growth and Biofilm Formation of Porphyromonas gingivalis and Prevotella intermedia","volume":"53","author":"Wakabayashi","year":"2009","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1139\/o06-053","article-title":"Effect of Lactoferrin on Enteroaggregative E. coli (EAEC)","volume":"84","author":"Ochoa","year":"2006","journal-title":"Biochem. Cell. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1007\/s10156-011-0287-1","article-title":"Inhibitory Effects of Lactoferrin on Biofilm Formation in Clinical Isolates of Pseudomonas aeruginosa","volume":"18","author":"Kamiya","year":"2012","journal-title":"J. Infect. Chemother."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2124","DOI":"10.1002\/cbic.202100016","article-title":"Antibacterial and Anti-Biofilm Activity of the Human Breast Milk Glycoprotein Lactoferrin against Group B Streptococcus","volume":"22","author":"Lu","year":"2021","journal-title":"ChemBioChem"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1023\/B:BIOM.0000027704.53859.d3","article-title":"Both Lactoferrin and Iron Influence Aggregation and Biofilm Formation in Streptococcus mutans","volume":"17","author":"Francesca","year":"2004","journal-title":"Biometals"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"891","DOI":"10.1007\/s10534-014-9752-5","article-title":"Effect of Bovine Apo-Lactoferrin on the Growth and Virulence of Actinobacillus pleuropneumoniae","volume":"27","year":"2014","journal-title":"BioMetals"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.jgar.2016.08.003","article-title":"Antibiofilm Effect of Warfarin on Biofilm Formation of Escherichia coli Promoted by Antimicrobial Treatment","volume":"7","author":"Ojima","year":"2016","journal-title":"J. Glob. Antimicrob. Resist."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1186\/s13567-020-00759-z","article-title":"Effect of Apo-Lactoferrin on Leukotoxin and Outer Membrane Vesicles of Mannheimia haemolytica A2","volume":"51","author":"Zenteno","year":"2020","journal-title":"Vet. Res."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Ram\u00edrez-Rico, G., Martinez-Castillo, M., Avalos-G\u00f3mez, C., and de la Garza, M. (2021). Bovine Apo-Lactoferrin Affects the Secretion of Proteases in Mannheimia haemolytica A2. Access Microbiol., 3.","DOI":"10.1099\/acmi.0.000269"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/j.cvfa.2010.10.016","article-title":"Treatment and Control of Respiratory Disease in Sheep","volume":"27","author":"Scott","year":"2011","journal-title":"Vet. Clin. North Am.\u2014Food Anim. Pract."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/j.vetmic.2016.11.012","article-title":"Mannheimia haemolytica Biofilm Formation on Bovine Respiratory Epithelial Cells","volume":"197","author":"Boukahil","year":"2016","journal-title":"Vet. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.chom.2019.06.002","article-title":"Surviving as a Community: Antibiotic Tolerance and Persistence in Bacterial Biofilms","volume":"26","author":"Yan","year":"2019","journal-title":"Cell Host Microbe"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Angulo-Zamudio, U.A., Vidal, J.E., Nazmi, K., Bolscher, J.G.M., Leon-Sicairos, C., Antezana, B.S., Canizalez-Roman, A., and Le\u00f3n-Sicairos, N. (2019). Lactoferrin Disaggregates Pneumococcal Biofilms and Inhibits Acquisition of Resistance Through Its DNase Activity. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.02386"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1080\/08927014.2013.773317","article-title":"Mini-Review: Lactoferrin: A Bioinspired, Anti-Biofilm Therapeutic","volume":"29","author":"Ammons","year":"2013","journal-title":"Biofouling"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1471","DOI":"10.1271\/bbb.62.1471","article-title":"Binding of Lactoferrin to Bacterial Cells of the Clostridium Species and Their Agglutination","volume":"62","author":"Tomita","year":"1998","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1701","DOI":"10.1007\/s42770-021-00624-x","article-title":"Microbial Biofilm: Formation, Architecture, Antibiotic Resistance, and Control Strategies","volume":"52","author":"Rather","year":"2021","journal-title":"Braz. J. Microbiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1236","DOI":"10.1128\/iai.62.4.1236-1240.1994","article-title":"Lactoferrin Binds to Porins OmpF and OmpC in Escherichia coli","volume":"62","author":"Erdei","year":"1994","journal-title":"Infect. Immun."},{"key":"ref_37","first-page":"1","article-title":"Two Outer Membrane Proteins Are Bovine Lactoferrin-Binding Proteins in Mannheimia haemolytica A1","volume":"47","year":"2016","journal-title":"Vet. Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1016\/j.ijantimicag.2008.08.013","article-title":"In Vitro Susceptibility of Established Biofilms Composed of a Clinical Wound Isolate of Pseudomonas aeruginosa Treated with Lactoferrin and Xylitol","volume":"33","author":"Ammons","year":"2009","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s11274-017-2334-2","article-title":"Killing Activity of LFchimera on Periodontopathic Bacteria and Multispecies Oral Biofilm Formation in Vitro","volume":"33","author":"Ruangcharoen","year":"2017","journal-title":"World J. Microbiol. Biotechnol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1111\/mmi.13698","article-title":"Biofilm Dispersal: Multiple Elaborate Strategies for Dissemination of Bacteria with Unique Properties","volume":"105","author":"Guilhen","year":"2017","journal-title":"Mol. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Zhao, A., Sun, J., and Liu, Y. (2023). Understanding Bacterial Biofilms: From Definition to Treatment Strategies. Front. Cell. Infect. Microbiol., 13.","DOI":"10.3389\/fcimb.2023.1137947"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Ram\u00edrez-Rico, G., Martinez-Castillo, M., Ruiz-Maz\u00f3n, L., Meneses-Romero, E.P., Palacios, J.A.F., D\u00edaz-Aparicio, E., Abascal, E.N., and de la Garza, M. (2024). Identification, Biochemical Characterization, and In Vivo Detection of a Zn-Metalloprotease with Collagenase Activity from Mannheimia haemolytica A2. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25021289"},{"key":"ref_43","first-page":"e2437","article-title":"Microtiter Dish Biofilm Formation Assay","volume":"47","year":"2010","journal-title":"J. Vis. Exp."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1150","DOI":"10.1007\/s00284-021-02373-4","article-title":"Biofilm Formation by Salmonella enterica Strains","volume":"78","author":"Shatila","year":"2021","journal-title":"Curr. Microbiol."},{"key":"ref_45","first-page":"A.3A.1","article-title":"Assays for Determination of Protein Concentration","volume":"2016","author":"Olson","year":"2016","journal-title":"Curr. Protoc. Pharmacol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Chaganti, L.K., Venkatakrishnan, N., and Bose, K. (2018). An Efficient Method for FITC Labelling of Proteins Using Tandem Affinity Purification. Biosci. Rep., 38.","DOI":"10.1042\/BSR20181764"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/16\/8718\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T15:34:15Z","timestamp":1760110455000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/16\/8718"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,8,9]]},"references-count":46,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2024,8]]}},"alternative-id":["ijms25168718"],"URL":"https:\/\/doi.org\/10.3390\/ijms25168718","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,8,9]]}}}