Skip to main content
SpringerLink
Log in

Biofilm formation and antibiotic susceptibility in dispersed cells versus planktonic cells from clinical, industry and environmental origins

  • Original Paper
  • Published:
Antonie van Leeuwenhoek Aims and scope Submit manuscript

Abstract

We examined the cell-surface physicochemical properties, the biofilm formation capability and the antibiotic susceptibility in dispersed cells (from an artificial biofilm of alginate beads) and compared with their planktonic (free-swimming) counterparts. The strains used were from different origins, such as clinical (Acinetobacter baumannii AB4), cosmetic industry (Klebsiella oxytoca EU213, Pseudomonas aeruginosa EU190), and environmental (Halomonas venusta MAT28). In general, dispersed cells adhered better to surfaces (measured as the “biofilm index”) and had a greater hydrophobicity [measured as the microbial affinity to solvents (MATS)] than planktonic cells. The susceptibility to two antibiotics (ciprofloxacin and tetracycline) of dispersed cells was higher compared with that of their planktonic counterparts (tested by the “bactericidal index”). Dispersed and planktonic cells exhibited differences in cell permeability, especially in efflux pump activity, which could be related to the differences observed in susceptibility to antibiotics. At 1 h of biofilm formation in microtiter plates, dispersed cells treated with therapeutic concentration of ciprofloxacin yielded a lower biofilm index than the control dispersed cells without ciprofloxacin. With respect to the planktonic cells, the biofilm index was similar with and without the ciprofloxacin treatment. In both cases there were a reduction of the number of bacteria measured as viable count of the supernatant. The lower biofilm formation in dispersed cells with ciprofloxacin treatment may be due to a significant increase of biofilm disruption with respect to the biofilm from planktonic cells. From a clinical point of view, biofilms formed on medical devices such as catheters, cells that can be related to an infection were the dispersed cells. Our results showed that early treatment with ciprofloxacin of dispersed cells could diminishe bacterial dispersion and facilitate the partial elimination of the new biofilm formed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Agwuh KN, MacGowan A (2006) Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines. J Antimicrob Chemother 58:256–265

    Article  CAS  PubMed  Google Scholar 

  • Ahamad PYA, Kunhi AAM (2011) Enhanced degradation of phenol by Pseudomonas sp. CP4 entrapped in agar and calcium alginate beads in batch and continuous processes. Biodegradation 22:253–265

    Article  Google Scholar 

  • Barros J, Grenho L, Manuel CM, Ferreira C, Melo LF, Nunes OC, Monteiro FJ, Ferraz MP (2013) A modular reactor to simulate biofilm development in orthopedic materials. Int Microbiol 16:191–198

    CAS  PubMed  Google Scholar 

  • Bazyleu A, Kumar A (2014) Incubation temperature, osmolarity, and salicylate affect the expression of resistance-nodulation-division efflux pumps and outer membrane porins in Acinetobacter baumannii ATCC19606T. FEMS Microbiol Lett 357:136–143

    CAS  PubMed  Google Scholar 

  • Bellon-Fontaine MN, Rault J, van Oss CJ (1996) Microbial adhesion to solvents: a novel method to determine the electron-donor/electron-acceptor or Lewis acid-base properties of microbial cells. Colloid Surf B 7:47–53

    Article  CAS  Google Scholar 

  • Berlanga M, Guerrero R (2016) Living together in biofilms: the microbial cell factory and its biotechnological implications. Microb Cell Fact 15:165. doi:10.1186/s12934-016-0569-5

    Article  PubMed  PubMed Central  Google Scholar 

  • Berlanga M, Viñas M (2000) Salicylate induction of phenotypic resistance to quinolones in Serratia marcescens. J Antimicrob Chemother 46:279–282

    Article  CAS  PubMed  Google Scholar 

  • Berlanga M, Ruiz N, Hernández-Borrell J, Montero T, Viñas M (2000) Role of the outer membrane in the accumulation of quinolones by Serratia marcescens. Can J Microbiol 46:716–722

    Article  CAS  PubMed  Google Scholar 

  • Berlanga M, Montero MT, Hernández-Borrell J, Viñas M (2004) Influence of the cell wall on ciprofloxacin susceptibility in selected wild-type Gram-negative and Gram-positive bacteria. Int J Antimicrob Agents 23:627–630

    Article  CAS  PubMed  Google Scholar 

  • Berlanga M, Miñana-Galbis D, Domènech Ò, Guerrero R (2012) Enhanced polyhydroxyalkanoates accumulation by Halomonas spp. in artificial biofilms of alginate beads. Int Microbiol 15:191–199

    CAS  PubMed  Google Scholar 

  • Berlanga M, Domènech Ò, Guerrero R (2014) Biofilm formation on polystyrene in detached vs. planktonic cells of polyhydroxyalkanoate-accumulation Halomonas venusta. Int Microbiol 17:205–212

    CAS  PubMed  Google Scholar 

  • Biniarz P, Baranowska G, Feder-Kubis J, Krasowska A (2015) The lipopeptides pseudofactin II and surfactin effectively decrease Candida albicans adhesion and hydrophobicity. Antonie van Leeuwenhoek 108:343–353

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Boonaert CJP, Dufrêne YF, Derclaye SR, Rouxhet PG (2001) Adhesion of Lactococcus lactis to model substrata: direct study of the interface. Colloid Surface B 22:171–182

    Article  CAS  Google Scholar 

  • Bos R, van der Mei HC, Busscher HJ (1999) Physico-chemistry of initial microbial adhesive interactions—its mechanisms and methods for study. FEMS Microbiol Rev 23:179–230

    Article  CAS  PubMed  Google Scholar 

  • Boy D, Well M, Kinzing-Schippers M, Sörgel F, Ankel-Fuchs D, Naver KD (2004) Urinary bactericidal activity, urinary excretion and plasma concentrations of gatifloxacin (400 mg) versus ciprofloxacin (500 mg) in healthy volunteers after a single oral dose. Int J Antimicrob Agents 23:S6–S16

    Article  CAS  PubMed  Google Scholar 

  • Bruchmann S, Dötsch A, Nouri B, Chaberny IF, Häussler S (2013) Quantitative contributions of target alteration and decreased drug accumulation to Pseudomonas aeruginosa fluoroquinolone resistance. Antimicrob Agents Chemother 57:1361–1368

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chagnot C, Zorgani MA, Astruc T, Desvaux M (2013) Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective. Front Microbiol 4:303. doi:10.3389/fmicb.2013.00303

    Article  PubMed  PubMed Central  Google Scholar 

  • Chen G, Walker SL (2007) Role of solution chemistry and ion valence on the adhesion kinetics of groundwater and marine bacteria. Langmuir 23:7162–7169

    Article  CAS  PubMed  Google Scholar 

  • Chia TWR, Fegan N, McMeekin TA, Dykes GA (2008) Salmonella Sofia differs from other poultry-associated Salmonella serovars with respect to cell surface hydrophobicity. J Food Protect 71:2421–2428

    Article  CAS  Google Scholar 

  • Chua SL, Liu Y, Yam JKH, Chen Y, Vejborg RM, Tan BGC, Kjelleberg S, Tolker-Nielsen T, Givskov M, Yang L (2014) Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles. Nat Commun 5:4462. doi:10.1038/ncomms5462

    Article  CAS  PubMed  Google Scholar 

  • De Angelis M, Siragusa S, Campanella D, Di Cagno R, Gobbetti M (2015) Comparative proteomic analysis of biofilm and planktonic cells of Lactobacillus plantarum DB200. Proteomics 15:2244–2257

    Article  PubMed  Google Scholar 

  • Delcour AH (2009) Outer membrane permeability and antibiotic resistance. Biochem Biophys Acta 1794:808–816

    CAS  PubMed  Google Scholar 

  • Djeribi R, Boucherit Z, Bouchloukh W, Zouaoui W, Latrache H, Hamadi F, Menaa B (2013) A study of pH effects on the bacterial surface physicochemical properties of Acinetobacter baumannii. Colloids Surface B 102:540–545

    Article  CAS  Google Scholar 

  • Donlan RM (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis 8:881–890

    Article  PubMed  PubMed Central  Google Scholar 

  • Giaouris E, Chapot-Chartier M-P, Briandet R (2009) Surface physicochemical analysis of natural Lactococcus lactis strains reveals the existence of hydrophobic and low charged strains with altered adhesive properties. Int J Food Microbiol 131:2–9

    Article  CAS  PubMed  Google Scholar 

  • Gilley RP, Orihuela CJ (2014) Pneumococci in biofilms are non-invasive: implications on nasopharyngeal colonization. Front Cell Infect Microbiol 4:163. doi:10.3389/fcimb.2014.00163

    Article  PubMed  PubMed Central  Google Scholar 

  • Gomes LC, Silva LN, Simões M, Melo LF, Mergulhão FJ (2014) Escherichia coli adhesion, biofilm development and antibiotic susceptibility on biomedical materials. J Biomed Mater Res 103:1414–1423

    Article  Google Scholar 

  • Guilhen C, Charbonnel N, Parisot N, Gueguen N, Iltis A, Forestier C, Balestrino D (2016) Transcriptional profiling of Klebsiella pneumoniae defines signatures for planktonic, sessile and biofilm-dispersed cells. BMC Genomics. doi:10.1186/s12864-016-2557-x

    PubMed  PubMed Central  Google Scholar 

  • He X, Lu F, Yuan F, Jiang D, Zhao P, Zhu J, Cheng H, Cao J, Guozhong L (2015) Biofilm formation caused by clinical Acinetobacter baumannii isolates is associated with overexpression of the AdeFGH efflux pump. Antimicrob Agents Chemother 59:4817–4825

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Horii T, Osaki M, Muramatsu H (2008) Fluoroquinolone resistance in clinical isolates of Klebsiella oxytoca. Chemotherapy 54:323–327

    Article  CAS  PubMed  Google Scholar 

  • Huang H-Y, Tang Y-J, King VA-E, Chou J-W, Tsen J-H (2015) Properties of Lactobacillus reuteri chitosan-calcium-alginate encapsulation under simulated gastrointestinal conditions. Int Microbiol 18:61–69

    CAS  PubMed  Google Scholar 

  • Hunt SM, Werner EM, Huang B, Hamilton MA, Stewart PS (2004) Hypothesis for the role of nutrient starvation in biofilm detachment. Appl Environ Microbiol 70:7418–7425

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jensen PØ, Givskov M, Bjamsholt T, Moser C (2010) The immune System vs Pseudomonas aeruginosa biofilms. FEMS Immunol Med Microbiol 59:292–305

    Article  CAS  PubMed  Google Scholar 

  • Junter G-A, Coquet L, Vilain S, Jouenne T (2002) Immobilized-cell physiology: current data and potentialities of proteomics. Enzyme Microb Technol 31:201–212

    Article  CAS  Google Scholar 

  • Katsikogianni M, Missirlis YF (2004) Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-material interactions. Eur Cells Mater 8:37–57

    Article  CAS  Google Scholar 

  • Knudsen GM, Nielsen M-B, Grassby T, Danino-Appleton V, Thomsen LE, Colquhoun IJ, Brocklehurst TF, Olsen JE, Hinton JCD (2012) A third mode of surface-associates growth: immobilization of Salmonella enterica serovar Thyphimurium modulates the RpoS-directed transcriptional programme. Environ Microbiol 14:1855–1875

    Article  CAS  PubMed  Google Scholar 

  • Koerdt A, Gödeke J, Berger J, Thormann KM, Albers S-J (2010) Crenarchaeal biofilm formation under extreme conditions. PLoS ONE 5:e14104. doi:10.1371/journal.pone.0014104

    Article  PubMed  PubMed Central  Google Scholar 

  • Kolter R (2010) Biofilms in lab and nature: a molecular geneticist’s voyage to microbial ecology. Int Microbiol 13:1–7

    PubMed  Google Scholar 

  • Krasowska A, Sigler K (2014) How microorganisms use hydrophobicity and what does this mean for human needs? Front Cell Infect Microbiol 4:112. doi:10.3389/fcimb.2014.00112

    Article  PubMed  PubMed Central  Google Scholar 

  • Lallemand EA, Lacroix MZ, Toutain P-L, Boullier S, Ferran AA, Bousquet-Melou A (2016) In vitro degradation of antimicrobials during use of broth microdilution method can increase the measured minimal inhibitory and micromal bactericidal concentrations. Front Microbiol. doi:10.3389/fmicb.2016.02051

    PubMed  PubMed Central  Google Scholar 

  • Liu J, Ling J-Q, Wu CD (2013) Physiological properties of Streptococcus mutants UA159 biofilm-detached cells. FEMS Microbiol Lett 340:11–18

    Article  CAS  PubMed  Google Scholar 

  • Lüdecke C, Jandt KD, Siegismund D, Kujau MJ, Zang E, Rettenmayr M, Bossert J, Roth M (2014) Reproducible biofilm cultivation of chemostat-grown Escherichia coli and investigation of bacterial adhesion on biomaterials using a non-constant-depth film fermenter. PLoS ONE 9:e84837. doi:10.1371/journal.pone.0084837

    Article  PubMed  PubMed Central  Google Scholar 

  • Ly MH, Vo NH, Le TM, Belin J-M, Waché Y (2006) Diversity of the surface properties of Lactococci and consequences on adhesion to food components. Colloids Surface B 52:149–153

    Article  CAS  Google Scholar 

  • McDougald D, Rice SA, Barraud N, Steinberg PD, Kjelleberg S (2012) Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal. Nat Rev Microbiol 10:39–50

    CAS  Google Scholar 

  • Monds RD, O’Toole GA (2009) The development model of microbial biofilms: ten years of a paradigm up for review. Trends Microbiol 17:73–87

    Article  CAS  PubMed  Google Scholar 

  • Morrissey I (1997) Bactericidal index: a new way to assess quinolone bactericidal activity in vitro. J Antimicrob Chemother 39:713–717

    Article  CAS  PubMed  Google Scholar 

  • Nielsen IE, Cars O, Friberg LE (2011) Pharmacokinetic/Pharmacodynamic (PK/PD) indices of antibiotics predicted by a semimechanistic PKPD model: a step toward model-based dose optimization. Antimicrob Agents Chemother 55:4619–4630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • O’Toole GA, Kolter R (1998) Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol Microbiol 28:449–461

    Article  PubMed  Google Scholar 

  • Orús P, Gomez-Perez L, Leranoz S, Berlanga M (2015) Increasing antibiotic resistance in preservative-tolerant bacterial strains isolated from cosmetic products. Int Microbiol 18:51–59

    PubMed  Google Scholar 

  • Pagès J-M, James CE, Winterhalter M (2008) The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria. Nat Rev Microbiol 6:893–903

    Article  PubMed  Google Scholar 

  • Palmer J, Flint S, Brooks J (2007) Bacterial cell attachment, the beginning of a biofilm. J Ind Microbiol Biotechnol 34:577–588

    Article  CAS  PubMed  Google Scholar 

  • Park AJ, Murphy K, Krieger JR, Brewer D, Taylor P, Habash M, Khursigara CM (2014) A temporal examination of the planktonic and biofilm proteome of whole cell Pseudomonas aeruginosa PAO1 using quantitative mass spectrometry. Mol Cell Proteomics 13:1095–1105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Perez F, Endimiani A, Ray AJ et al (2010) Carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination. J Antimicrob Chemother. doi:10.1093/jac/dkq191

    PubMed Central  Google Scholar 

  • Peter H, Ylla I, Gudasz C, Romani AM, Sabater S, Tranvik LJ (2011) Multifunctionality and diversity in bacterial biofilms. PLoS ONE 6:e23225. doi:10.1371/journal.pone.0023225

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Petrova OE, Sauer K (2016) Escaping the biofilm in more than one way: desorption, detachment or dispersion. Curr Opin Microbiol 30:67–78

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Post DMB, Held JM, Ketterer MR, Philips NJ, Sahu A, Apicella MA, Gibson BW (2014) Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry. BMC Microbiol. doi:10.1186/s12866-014-0329-9

    PubMed  PubMed Central  Google Scholar 

  • Prada-López I, Quintas V, Casares-De-Cal MA, Suárez-Quintanilla JA, Suárez-Quintanilla D, Tomás I (2015) Ex vivo vs. in vivo antibacterial activity of two antiseptics on oral biofilm. Front Microbiol 6:655. doi:10.3389/fmicb.2015.00655

    Article  PubMed  PubMed Central  Google Scholar 

  • Preedy E, Perni S, Nipiĉ D, Bohinc K, Prokopovich P (2014) Surface roughness mediated adhesion forces between borosilicate glass and Gram-positive bacteria. Langmuir 30:9466–9476

    Article  CAS  PubMed  Google Scholar 

  • Rollet C, Gal L, Guzzo J (2009) Biofilm-detached cells, a transition from a sessile to planktonic phenotype: a comparative study of adhesion and physiological characteristics in Pseudomonas aeruginosa. FEMS Microbiol Lett 290:135–142

    Article  CAS  PubMed  Google Scholar 

  • Rumbo-Feal S, Gómez MJ, Gayoso C et al (2013) Whole transcriptome analysis of Acinetobacter baumannii assessed by RNA-sequencing reveals different mRNA expression profiles in Biofilm compared to planktonic cells. PLoS ONE. doi:10.1371/journal.pone.0072968

    PubMed  PubMed Central  Google Scholar 

  • Sachs JL, Hollowed AC (2012) The origins of cooperative bacterial communities. mBio 3:e00099–12. doi:10.1128/mBio.00099-12

    Article  PubMed  PubMed Central  Google Scholar 

  • Selimoglu SM, Elibol M (2010) Alginate as an immobilization material for Mab production via encapsulated hybridoma cells. Crit Rev Biotechnol 30:145–159

    Article  CAS  PubMed  Google Scholar 

  • Seper A, Pressler K, Kariisa A, Haid AG, Roier S, Leitner DR, Reidl J, Tamayo R, Schild S (2014) Identification of genes induced in Vibrio cholera in a dynamic biofilm system. Int J Med Microbiol 304:749–763

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Silva-Dias A, Miranda IM, Branco J, Monteiro-Soares M, Pina-Vaz C, Rodrigues AG (2015) Adhesion, biofilm formation, cell surface hydrophobicity, and antifungal planktonic susceptibility: relationship among Candida spp. Front Microbiol 6:205. doi:10.3389/fmicb.2015.00205

    Article  PubMed  PubMed Central  Google Scholar 

  • Simões LC, Simões M, Vieira MJ (2010) Adhesion and biofilm formation on polystyrene by drinking water-isolated bacteria. Antonie Van Leeuwenhoek 98:317–329

    Article  PubMed  Google Scholar 

  • Thormann KM, Saville RM, Shukla S, Spormann AM (2005) Induction of rapid detachment in Shewanella oneidensis MR-1 biofilms. J Bacteriol 187:1014–1021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tokunaga H, Mitsuo K, Kamekura M, Tokunaga M (2004) Major outer membrane proteins in moderately halophilic eubacteria of genera Chromohalobacter and Halomonas. J Basic Microbiol 44:232–240

    Article  CAS  PubMed  Google Scholar 

  • Trémoulet F, Duché O, Namane A, Martinie B (2002) European Listeria Genome Consortium, Labadie JC. Comparison of protein patterns of Listeria monocytogenes grown in biofilm or in planktonic mode by proteomic analysis. FEMS Microbiol Lett 210:25–31

    Article  PubMed  Google Scholar 

  • Uppuluri P, Chaturvedi AK, Srinivasan A et al (2010) Dispersion as an important step in the Candida albicans biofilm developmental cycle. PLoS Pathog 6:e1000828

    Article  PubMed  PubMed Central  Google Scholar 

  • Van der Werf MJ, Pieterse B, van Luijk N, Schuren F, van der Werff-van der Vat B, Overkamp K, Jellema RH (2006) Multivariate analysis of microarray data by principal component discriminant analysis: prioritizing relevant transcripts linked to the degradation of different carbohydrates in Pseudomonas putida S12. Microbiology 152:257–272

    Article  PubMed  Google Scholar 

  • Van Gestel J, Vlamakis H, Kolter R (2015) Division of labor in biofilms: the ecology of cell differentiation. Microbiol Spectrum. doi:10.1128/microbiolspec.MB-0002-2014

    Google Scholar 

  • Van Merode AEJ, van der Mei HC, Busscher HJ, Krom BP (2006) Influence of culture heterogeneity in cell surface charge on adhesion and biofilm formation by Enterococcus faecalis. J Bacteriol 188:2421–2426

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang Y-W, Prabhu P (2010) Alginate immobilization of recombinant Escherichia coli whole cells harboring l-arabinose isomerase for l-ribulose production. Bioprocess Biosyst Eng 33:741–748

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This study was funded by Department of Biology, Health and Environment, Section of Microbiology to MB.

Author information

Authors and Affiliations

  1. Department of Biology, Environment and Health, Section Microbiology, Faculty of Pharmacy and Food Sciences, University of Barcelona, Av. Joan XXIII 27-31, 08028, Barcelona, Spain

    Mercedes Berlanga & Laura Gomez-Perez

  2. School of Biological Sciences, University of East Anglia, Norwich, UK

    Laura Gomez-Perez

  3. Laboratory of Molecular Microbiology and Antimicrobials, Department of Pathology and Experimental Therapeutics, Faculty of Medicine, University of Barcelona, IDIBELL, Barcelona, Spain

    Ricardo Guerrero

  4. Barcelona Knowledge Hub, Academia Europaea, Barcelona, Spain

    Ricardo Guerrero

Authors
  1. Mercedes Berlanga
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laura Gomez-Perez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ricardo Guerrero
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mercedes Berlanga.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Berlanga, M., Gomez-Perez, L. & Guerrero, R. Biofilm formation and antibiotic susceptibility in dispersed cells versus planktonic cells from clinical, industry and environmental origins. Antonie van Leeuwenhoek 110, 1691–1704 (2017). https://doi.org/10.1007/s10482-017-0919-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10482-017-0919-2

Keywords

Search

Navigation