{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T07:20:40Z","timestamp":1792653640715,"version":"4.5.0"},"reference-count":57,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,9,18]],"date-time":"2021-09-18T00:00:00Z","timestamp":1631923200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004281","name":"Narodowe Centrum Nauki","doi-asserted-by":"publisher","award":["2016\/23\/B\/NZ7\/03236"],"award-info":[{"award-number":["2016\/23\/B\/NZ7\/03236"]}],"id":[{"id":"10.13039\/501100004281","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Streptococcus agalactiae is a relevant cause of neonatal mortality. It can be transferred to infants via the vaginal tract and cause meningitis, pneumonia, arthritis, or sepsis, among other diseases. The cause of therapy ineffectiveness and infection recurrence is the growth of bacteria as biofilms. To date, several research teams have attempted to find a suitable medium for the cultivation of S. agalactiae biofilms. Among others, simulated vaginal fluid has been used; however, biofilm production in this medium has been found to be lower than that in tryptic soy broth. We have previously shown that S. agalactiae can be successfully eradicated by photoinactivation in planktonic culture, but there have been no studies on biofilms. The aim of this study was to optimize S. agalactiae biofilm culture conditions to be used in photoinactivation studies. We compared biofilm production by four strains representing the most common serotypes in four different broth media with crystal violet staining. Then, we evaluated stationary biofilm culture in microtiter plates and biofilm growth in a CDC Biofilm Reactor\u00ae (BioSurface Technologies, Bozeman, MT, USA) under continuous flow conditions. Subsequently, we applied Rose Bengal-mediated photoinactivation to both biofilm models. We have shown that photoinactivation is efficient in biofilm eradication and is not cyto\/phototoxic to human keratinocytes. We found conditions allowing for stable and repetitive S. agalactiae biofilm growth in continuous flow conditions, which can be successfully utilized in photoinactivation assays and potentially in all other antibacterial studies.<\/jats:p>","DOI":"10.3390\/pathogens10091212","type":"journal-article","created":{"date-parts":[[2021,9,21]],"date-time":"2021-09-21T22:34:01Z","timestamp":1632263641000},"page":"1212","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Optimization of Streptococcus agalactiae Biofilm Culture in a Continuous Flow System for Photoinactivation Studies"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5548-8216","authenticated-orcid":false,"given":"Michal K.","family":"Pieranski","sequence":"first","affiliation":[{"name":"Laboratory of Photobiology and Molecular Diagnostics, Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, 80-307 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michal","family":"Rychlowski","sequence":"additional","affiliation":[{"name":"Laboratory of Virus Molecular Biology, Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, 80-307 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4041-0891","authenticated-orcid":false,"given":"Mariusz","family":"Grinholc","sequence":"additional","affiliation":[{"name":"Laboratory of Photobiology and Molecular Diagnostics, Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, 80-307 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,9,18]]},"reference":[{"key":"ref_1","first-page":"141","article-title":"Neonatal & maternal group B streptococcal infections: A comprehensive review","volume":"120","author":"Shet","year":"2004","journal-title":"Indian J. Med. Res."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Kaminska, D., Ratajczak, M., Szuma\u0142a-K\u0105kol, A., Dlugaszewska, J., Nowak-Malczewska, D.M., and Gajecka, M. (2020). Increasing resistance and changes in distribution of serotypes of Streptococcus agalactiae in poland. Pathogens, 9.","DOI":"10.3390\/pathogens9070526"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2055","DOI":"10.1128\/JCM.00154-09","article-title":"Diversity of group B streptococcus serotypes causing urinary tract infection in adults","volume":"47","author":"Ulett","year":"2009","journal-title":"J. Clin. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1093\/cid\/civ353","article-title":"Biofilms: An Underappreciated Mechanism of Treatment Failure and Recurrence in Vaginal Infections","volume":"61","author":"Muzny","year":"2015","journal-title":"Clin. Infect. Dis."},{"key":"ref_5","first-page":"86","article-title":"Biofilm bacteria: Formation and comparative susceptibility to antibiotics","volume":"66","author":"Olson","year":"2002","journal-title":"Can. J. Vet. Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2176","DOI":"10.1128\/AEM.03627-13","article-title":"Acidic pH strongly enhances in vitro biofilm formation by a subset of hypervirulent ST-17 Streptococcus agalactiae strains","volume":"80","author":"Martinelli","year":"2014","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1007\/s10482-011-9666-y","article-title":"Survival and biofilm formation by Group B streptococci in simulated vaginal fluid at different pHs","volume":"101","author":"Borges","year":"2012","journal-title":"Antonie Van Leeuwenhoek"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1007\/s00203-019-01727-8","article-title":"Biofilm production under different atmospheres and growth media by Streptococcus agalactiae isolated from milk of cows with subclinical mastitis","volume":"202","author":"Bonsaglia","year":"2020","journal-title":"Arch. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"103820","DOI":"10.1016\/j.micpath.2019.103820","article-title":"Vaginal dysbiotic bacteria act as pathobionts of the protozoal pathogen Trichomonas vaginalis","volume":"138","author":"Hinderfeld","year":"2020","journal-title":"Microb. Pathog."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e9917","DOI":"10.7717\/peerj.9917","article-title":"Evaluation of different culture media to support in vitro growth and biofilm formation of bacterial vaginosis-associated anaerobes","volume":"8","author":"Rosca","year":"2020","journal-title":"PeerJ"},{"key":"ref_11","first-page":"101","article-title":"Lysozyme as a cotreatment during antibiotics use against vaginal infections: An in vitro study on gardnerella vaginalis biofilm models","volume":"19","author":"Thellin","year":"2016","journal-title":"Int. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12866-020-01753-3","article-title":"Lactobacilli spp.: Real-time evaluation of biofilm growth","volume":"20","author":"Martinez","year":"2020","journal-title":"BMC Microbiol."},{"key":"ref_13","first-page":"1","article-title":"Effect of 17\u03b2-estradiol on a human vaginal Lactobacillus crispatus strain","volume":"11","author":"Clabaut","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"12004","DOI":"10.3390\/ijms140612004","article-title":"Interactions between Lactobacillus crispatus and bacterial vaginosis (BV)-associated bacterial species in initial attachment and biofilm formation","volume":"14","author":"Machado","year":"2013","journal-title":"Int. J. Mol. Sci."},{"key":"ref_15","first-page":"617","article-title":"Factors Determining the Susceptibility of Bacteria to Antibacterial Photodynamic Inactivation","volume":"8","author":"Michalska","year":"2021","journal-title":"Front. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.pdpdt.2017.11.012","article-title":"Antimicrobial photodynamic activity of Rose Bengal, alone or in combination with Gentamicin, against planktonic and biofilm Staphylococcus aureus","volume":"21","author":"Ballesta","year":"2018","journal-title":"Photodiagn. Photodyn. Ther."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Silva, A.F., Borges, A., Freitas, C.F., Hioka, N., Mikcha, J.M.G., and Sim\u00f5es, M. (2018). Antimicrobial Photodynamic Inactivation Mediated by Rose Bengal and Erythrosine Is Effective in the Control of Food-Related Bacteria in Planktonic and Biofilm States. Molecules, 23.","DOI":"10.3390\/molecules23092288"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/1010-6030(89)85002-6","article-title":"Rose Bengal","volume":"47","author":"Neckers","year":"1989","journal-title":"J. Photochem. Photobiol. A Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-020-69403-2","article-title":"Photodynamic exposure of Rose-Bengal inhibits Tau aggregation and modulates cytoskeletal network in neuronal cells","volume":"10","author":"Dubey","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1016\/j.freeradbiomed.2020.09.003","article-title":"Increased photoinactivation stress tolerance of Streptococcus agalactiae upon consecutive sublethal phototreatments","volume":"160","author":"Pieranski","year":"2020","journal-title":"Free Radic. Biol. Med."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Shang, F., Wang, H., and Xue, T. (2020). Anti-biofilm effect of tea saponin on a Streptococcus agalactiae strain isolated from bovine mastitis. Animals, 10.","DOI":"10.3390\/ani10091713"},{"key":"ref_22","first-page":"1","article-title":"Antimicrobial and antibiofilm potentials of cinnamon oil and silver nanoparticles against Streptococcus agalactiae isolated from bovine mastitis: New avenues for countering resistance","volume":"17","author":"Ammar","year":"2021","journal-title":"BMC Vet. Res."},{"key":"ref_23","first-page":"861497","article-title":"Effect of eugenol against streptococcus agalactiae and synergistic interaction with biologically produced silver nanoparticles","volume":"2015","author":"Morey","year":"2015","journal-title":"Evid.-Based Complement. Altern. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e16058","DOI":"10.5812\/jjm.16058","article-title":"Effects of Benzalkonium Chloride on Planktonic Growth and Biofilm Formation by Animal Bacterial Pathogens","volume":"8","author":"Ebrahimi","year":"2015","journal-title":"Jundishapur J. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1038\/s41429-019-0151-6","article-title":"1-Amino-2\u2032-fucosyllactose inhibits biofilm formation by Streptococcus agalactiae","volume":"72","author":"Craft","year":"2019","journal-title":"J. Antibiot. (Tokyo)"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1021\/acsinfecdis.7b00183","article-title":"Antimicrobial and Antibiofilm Activity of Human Milk Oligosaccharides against Streptococcus agalactiae, Staphylococcus aureus, and Acinetobacter baumannii","volume":"4","author":"Ackerman","year":"2018","journal-title":"ACS Infect. Dis."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1016\/j.resmic.2020.08.001","article-title":"Biofilm formation by ST17 and ST19 strains of Streptococcus agalactiae","volume":"171","author":"Silvestre","year":"2020","journal-title":"Res. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1111\/lam.13015","article-title":"Biofilm formation on different pH conditions by Streptococcus agalactiae isolated from bovine mastitic milk","volume":"67","author":"Miranda","year":"2018","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"9923","DOI":"10.1002\/chem.202000354","article-title":"Synthetic Ellagic Acid Glycosides Inhibit Early Stage Adhesion of Streptococcus agalactiae Biofilms as Observed by Scanning Electron Microscopy","volume":"26","author":"Chambers","year":"2020","journal-title":"Chem.-A Eur. J."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1007\/s10482-012-9736-9","article-title":"The impact of pH and nutrient stress on the growth and survival of streptococcus agalactiae","volume":"102","author":"Yang","year":"2012","journal-title":"Antonie Van Leeuwenhoek."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"374","DOI":"10.4172\/2157-7579.1000374","article-title":"Antimicrobial Resistance of Biofilm-Forming Streptococcus agalactiae Isolated from Bovine Mastitis","volume":"7","author":"Boonyayatra","year":"2016","journal-title":"J. Vet. Sci. Technol."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Shan, Y., Yang, N., Teng, D., Wang, X., Mao, R., Hao, Y., Ma, X., Fan, H., and Wang, J. (2020). Recombinant of the staphylococcal bacteriophage lysin CHAPk and its elimination against streptococcus agalactiae biofilms. Microorganisms, 8.","DOI":"10.3390\/microorganisms8020216"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"252","DOI":"10.1016\/j.colsurfb.2017.10.050","article-title":"In vitro anti-biofilm activity of a biphasic gentamicin-loaded calcium sulfate\/hydroxyapatite bone graft substitute","volume":"161","author":"Butini","year":"2018","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3085","DOI":"10.1093\/jac\/dkx265","article-title":"Synergistic antibiotic activity against planktonic and biofilmembedded Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis","volume":"72","author":"Moreno","year":"2017","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1016\/j.mimet.2007.04.010","article-title":"Comparative evaluation of biofilm disinfectant efficacy tests","volume":"70","author":"Goeres","year":"2007","journal-title":"J. Microbiol. Methods"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1186\/1471-2180-11-75","article-title":"The impact of the competence quorum sensing system on Streptococcus pneumoniae biofilms varies depending on the experimental model","volume":"11","author":"Trappetti","year":"2011","journal-title":"BMC Microbiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1016\/j.msec.2015.05.036","article-title":"Silver nanoparticles with antimicrobial activities against Streptococcus mutans and their cytotoxic effect","volume":"55","author":"Boegli","year":"2015","journal-title":"Mater. Sci. Eng. C"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"150653","DOI":"10.1155\/2013\/150653","article-title":"Innovative strategies to overcome biofilm resistance","volume":"2013","author":"Taraszkiewicz","year":"2013","journal-title":"Biomed. Res. Int."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e154","DOI":"10.1111\/j.1439-0507.2010.01862.x","article-title":"Effect of serum and surface characteristics on Candida albicans biofilm formation","volume":"54","author":"Frade","year":"2011","journal-title":"Mycoses"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"908","DOI":"10.1111\/jam.12272","article-title":"Adherence and biofilm formation of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium abscessus to household plumbing materials","volume":"115","author":"Mullis","year":"2013","journal-title":"J. Appl. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"734459","DOI":"10.1016\/j.aquaculture.2019.734459","article-title":"Formation of biofilms of the salmon pathogen Flavobacterium psychrophilum in differents surfaces using the CDC biofilm reactor","volume":"514","author":"Vidal","year":"2020","journal-title":"Aquaculture"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Williams, D.L., Smith, S.R., Peterson, B.R., Allyn, G., Cadenas, L., Epperson, R.T., and Looper, R.E. (2019). Growth substrate may influence biofilm susceptibility to antibiotics. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0206774"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"3305","DOI":"10.1007\/s11999-014-3538-5","article-title":"Biofilm Growth Has a Threshold Response to Glucose In Vitro","volume":"472","author":"Waldrop","year":"2014","journal-title":"Clin. Orthop. Relat. Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1081\/FBT-120019984","article-title":"Medium Evaluation and Plastic Composite Support Ingredient Selection for Biofilm Formation and Succinic Acid Production by Actinobacillus succinogenes","volume":"17","author":"Urbance","year":"2003","journal-title":"Food Biotechnol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1111\/j.1574-6968.2011.02492.x","article-title":"TcyR regulates l-cystine uptake via the TcyABC transporter in Streptococcus mutans","volume":"328","author":"Kim","year":"2012","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1099\/jmm.0.46844-0","article-title":"Evaluation of different iron sources and their influence in biofilm formation by the dental pathogen Actinobacillus actinomycetemcomitans","volume":"56","author":"Rhodes","year":"2007","journal-title":"J. Med. Microbiol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"239","DOI":"10.4315\/0362-028X.JFP-12-321","article-title":"Influence of Glucose Concentrations on Biofilm Formation, Motility, Exoprotease Production, and Quorum Sensing in Aeromonas hydrophila","volume":"76","author":"Jahid","year":"2013","journal-title":"J. Food Prot."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"443","DOI":"10.4155\/fmc-2018-0329","article-title":"Photoinactivation of ESKAPE pathogens: Overview of novel therapeutic strategy","volume":"11","author":"Nakonieczna","year":"2019","journal-title":"Future Med. Chem."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"118","DOI":"10.2478\/s11536-011-0107-0","article-title":"Photodynamic therapy (PDT) for disinfection of oral wounds. In vitro study","volume":"7","year":"2012","journal-title":"Open Med."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1016\/j.pdpdt.2015.08.007","article-title":"In vitro photoinactivation of bovine mastitis related pathogens","volume":"13","author":"Sellera","year":"2016","journal-title":"Photodiagn. Photodyn. Ther."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"715565","DOI":"10.3389\/fchem.2021.715565","article-title":"Efficient Inhibition of Streptococcus agalactiae by AIEgen-Based Fluorescent Nanomaterials","volume":"9","author":"Yi","year":"2021","journal-title":"Front. Chem."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"111996","DOI":"10.1016\/j.jphotobiol.2020.111996","article-title":"The importance of porphyrins in blue light suppression of Streptococcus agalactiae","volume":"212","author":"Bumah","year":"2020","journal-title":"J. Photochem. Photobiol. B Biol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"112149","DOI":"10.1016\/j.jphotobiol.2021.112149","article-title":"Blue light absorbing pigment in Streptococcus agalactiae does not potentiate the antimicrobial effect of pulsed 450 nm light","volume":"216","author":"Bumah","year":"2021","journal-title":"J. Photochem. Photobiol. B Biol."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Ohtsu, I., Kawano, Y., Suzuki, M., Morigasaki, S., Saiki, K., Yamazaki, S., Nonaka, G., and Takagi, H. (2015). Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0120619"},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Bloom, S.M., Mafunda, N.A., Woolston, B.M., Hayward, M.R., Frempong, J.F., Abai, A.B., Xu, J., Mitchell, A.J., Westergaard, X., and Hussain, F.A. (2021). Cysteine dependence in Lactobacillus iners constitutes a novel therapeutic target to modify the vaginal microbiota. bioRxiv.","DOI":"10.1101\/2021.06.12.448098"},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Wo\u017aniak, A., Kruszewska, B., Piera\u0144ski, M.K., Rych\u0142owski, M., and Grinholc, M. (2021). Antimicrobial Photodynamic Inactivation Affects the Antibiotic Susceptibility of Enterococcus spp. Clinical Isolates in Biofilm and Planktonic Cultures. Biomolecules, 11.","DOI":"10.3390\/biom11050693"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"1949","DOI":"10.3389\/fmicb.2018.01949","article-title":"Rose Bengal-Mediated Photoinactivation of Multidrug Resistant Pseudomonas aeruginosa Is Enhanced in the Presence of Antimicrobial Peptides","volume":"9","author":"Nakonieczna","year":"2018","journal-title":"Front. Microbiol."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/10\/9\/1212\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:01:40Z","timestamp":1760166100000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/10\/9\/1212"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,9,18]]},"references-count":57,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2021,9]]}},"alternative-id":["pathogens10091212"],"URL":"https:\/\/doi.org\/10.3390\/pathogens10091212","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,9,18]]}}}