/ J of IMAB. 2015, vol. 21, issue 2/ http://www.journal-imab-bg.org 793

ADHESION AND SURFACE GROWTH OFSTAPHYLOCOCCUS AUREUS ANDLACTOBACILLUS PLANTARUM ON VARIOUSMETALS

Tsveteslava V. Ignatova-Ivanova, Sevginar F. Ibrjam, Ismail E. Ismailov, ValerijCh. Christov, Radoslav I. Ivanov.Department of Biology, Faculty of Natural Sciences, University of Shumen,Bulgaria.

Journal of IMAB - Annual Proceeding (Scientific Papers) 2015, vol. 21, issue 2Journal of IMABISSN: 1312-773Xhttp://www.journal-imab-bg.org

ABSTRACT:Background: One of the major drawbacks in the use

of biomedical materials is the occurrence of biomaterial-centered infections. Adhesion of microorganisms to animplant is mediated by their physico-chemical surfaceproperties and the properties of the biomaterial surfaceitself. Subsequent surface growth of the microorganisms willlead to a mature biofilm and infection, which is difficult toeradicate by antibiotics.

Objective: The purpose of this research is to examinethe adhesion in the combined cultivation of Staphylococcusaureus and the Lactobacillus plantarum probiotic bacteriumon the surface of different metals (copper, aluminium, low-carbon steel, and zinc).

Methods: The precise weighing (with an allowanceof 0,0001 g) of the metal plates before and after thetreatment found a minimum negative change in their weight,which may be caused by reduction resulting from corrosionprocesses, on one hand, or growth because of the formingof a biofilm, on the other. The structure of the layer overthe metal plates was analysed by SEM (scanning electronmicroscopy) JSM 5510.

Results: The thinnest biofilm for both bacteria wasregistered on the surface of the copper plate. When acombined culture is used on the surface of the aluminiumand the steel plates, the pathogenic bacterium is adheredpredominantly. On the zinc plate it is only the probioticbacterium that adheres.

Conclusion: This is an initial research on thisproblem of significance for the doctors and it is about tobe further examined.

Keywords: adhesion, biofilm, Staphylococcusaureus, Lactobacillus plantarum

INTRODUCTION:Foreign materials are used more and more in modern

medicine after trauma, oncological surgery or wear toreplace, support or restore human body function, forexample in hip prostheses, prosthetic heart valves orcatheters [1]. The extended use of these materials, usuallyreferred to as biomaterials, has some major drawbacks. Oneof these is the possible occurrence of biomaterial-centeredinfections (BCI) [2]. The incidence of this type of infections

varies from 4% for hip prostheses [3] to 100% for urinarytract catheters after 3 weeks use [4]. Adhesion ofmicroorganisms to an implant is mediated by their physico-chemical surface properties and the properties of thebiomaterial surface itself. Subsequent surface growth of themicroorganisms will lead to a mature biofilm and infection,which is difficult to eradicate by antibiotics. treatment ofBCI is complicated, as microorganisms in a biofilm aremore resistant to antibiotics [5] than their planktoniccounterparts [6]. As a consequence and if possible, the onlyremedy is removal of the infected implant at the expenseof considerable costs and patient’s suffering. A moreconvenient way to deal with this problem is to prevent thedevelopment of an infectious biofilm on the biomaterialsurface. To achieve this, a thorough understanding of howthese biofilms develop is necessary [1]. The search forbiomaterials that are able to provide for the optimalresistance to the infection can be based only on the deepunderstanding of the interactions between bacteria andbiomaterials.

The purpose of this research is to examine theadhesion in vitro in the combined cultivation ofStaphylococcus aureus and the Lactobacillus plantarumprobiotic bacterium on the surface of different metals(copper, aluminium, low-carbon steel, and zinc).

MATERIALS AND METHODS:Test MicroorganismsStaphylococcus aureus 745 were obtained from the

Collection of the Department of General and AppliedMicrobiology, Sofia University. The isolate were checkedfor purity and maintained in slant of Nutrient agar. NutrientAgar (Biolife 272-20128, Milano, Italia) was the mediumused as the growth medium for the microbe.

The Lactobacillus plantarum strain was isolated fromcommercial probiotic product. The strain cultivated in mediaof MRS (de Mann Rogosa Sharpe, Biolife 272-20128,Milano, Italia) in composition, per liter: glucose – 20.0;Tween 80 - 1; pepton from casein - 10.0; meat extract - 8.0;yeast extract - 4.0; K2HPO4 - 2.0; sodium acetate - 5.0;amonium citrate - 2.0; MgSO4.7H2O - 0.2 and MnSO4 -0.05 g/L. The pH of media was adjusted to 6.5 with 1MNaOH. The basic media was sterilized by autoclaving at121°C for 20 min.

http://dx.doi.org/10.5272/jimab.2015212.793

794 http://www.journal-imab-bg.org / J of IMAB. 2015, vol. 21, issue 2/

Before the assays, the strains L. plantarum and S.aureus 745 were twice pre-cultured in MRS broth andNutrient broth respectively, for 24 h at 37° C. Exponentialcultures in broths were used as inoculum for the adhesionanalysis.

Preparation of the metal samplesThe steel plates made of copper, aluminium, low-

carbon steel, and zinc are weighed with an allowance of0,0001 g with an assay-balance. The precise weighing (withan allowance of 0,0001 g) of the metal plates before and afterthe treatment found a minimum negative change in theirweight, which may be caused by reduction resulting fromcorrosion processes, on one hand, or growth because of theforming of a biofilm, on the other. They are put sterilely in aliquid ambient which contains a mixture of L. plantarum and

S. aureus 745 in a proportion 1:1. The samples wereincubated at 37° C for 24 h. The structure of the layer overthe metal plates was analysed by SEM (scanning electronmicroscopy) JSM 5510.

All experiments were performed in triplicate.

RESULTS:The results obtained from the SEM analysis of the

adhesion ability of the tested microorganisms on the differentmetals are shown in figure 1. The thinnest biofilm for bothbacteria was registered on the surface of the copper plate (fig.1 B). When a combined culture is used on the surface of thealuminium and the steel plates, the pathogenic bacterium isadhered predominantly (fig.1 A and C). On the zinc plate itis only the probiotic bacterium that adheres (fig.1 D).

Fig. 1. SEM of the tested samples.

1C) aluminium; 1D) zinc.

1A) low-carbon steel; 1B) copper;

/ J of IMAB. 2015, vol. 21, issue 2/ http://www.journal-imab-bg.org 795

Van Loosdrecht et al. [7] concluded that adhesion ofbacteria does not directly influence their metabolism andgrowth yield. Changes in growth rate due to adhesion ofbacteria were suggested to be mainly the result of changesin nutrient availability [1]. Depending on the amount ofadsorbed nutrients and whether adsorption is easilyreversed, growth rates of adhering bacteria can be decreasedor increased with respect to the growth of their planktoniccounterparts. Probably the materials differences played arole. S. aureus grew faster on the metal, while S. epidermidisgrew faster on polymeric biomaterials [1].

DISCUSSION:When microorganisms have reached the biomaterial

surface, initial microbial adhesion can occur. Microbialadhesion is mediated by specific interactions between cellsurface structures

and specific molecular groups on the substratumsurface [8], or when viewed from an overall, physico-chemical view-point by non-specific interaction forces,including Lifshitz- Van der Waals forces, electrostatic forces,acid-base interactions and Brownian motion forces [9].

After adhesion to biomaterials most microorganismsstart secreting slime and embed themselves in a slime layer,the glycocalix, which is an important virulence factor forBCI and which explains the extraordinary prevalence ofslime producing S. epidermidis in BCI [8]. The glycocalixprovides protection against humoral and excreted cellularimmune components, as these can not readily diffusethrough the slime layer [5] and once a glycocalix hasformed a BCI with all its complications, includingultimately removal of the implant, seems almost inevitable.

In previous publications we have shown that lacticacid bacteria are capable of forming a biofilm by producingexopolysaccharides which protect the metals from corrosion[10, 11, 12]. Moreover, we have shown that some of the endproducts of the fermentation process are also able to forma protective layer on the metal surfaces [13]. However, whyit is only on the zinc plate that a biofilm of the beneficialbacteria is formed is a question difficult to answer at thisstage. In our opinion, differences are also likely to appearin the adhesion process under in vitro and in vivo conditionsbecause other processes are going to have an impact in theliving organism, too. Kristopher P. et.al. [14] concluded thathydrophobic and photoinduced superhydrophilic surface

coatings both have potential as a means of reducingmicrobial fouling of surfaces. According to the updatedparadigm for biocompatibility, as redrawn by Williams [15],a biomaterial should perform its designed function elicitingthe most appropriate tissue response. This performance isespecially expected now that biomaterials need to be ableto act as sophisticated gene/cell/drug delivery systems, andare more and more turned towards biotechnology and tissueengineering applications. Nevertheless, in the vast majorityof clinical implantations, the first requirement of a material’sbiocompatibility is that, whatever the desired function, thematerial shall not induce any adverse effects in the patient,“just as the first principle of Hippocrates was that the doctorshould do no harm” Biomaterial science has greatlyprogressed in the achievement of a safe biocompatibility,free from damage to neighbouring tissues. Unfortunately,as Gristina warned ever since 1987, a major drawback toimplanted devices remains “the possibility of bacterialadhesion to biomaterials, which causes biomaterial-centredinfection” [15].

CONCLUSION/S/:The various metabolic ways and the various end

metabolic products of the two types of bacteria,Staphylococcus aureus and Lactobacillus plantarum, couldexplain to a certain extent the different biofilms formed onthe different metal surfaces. Different types of complexcompounds are probably formed between the secretedexopolysaccharides or the end metabolic products and themetal surfaces. The mechanism of this process is still to beexplained. The adhesion of microbes to surfaces can beaffected by numerous physico-chemical factors, and thecomplexity of microbial adhesion has been demonstrated.There is no one clear explanation for the behaviour of allof the materials with regard to adhesion of microbes to theirsurface. Anti-infective biomaterials need to be tailoredaccording to the specific clinical application. All theirproperties have to be tuned to achieve the best anti-infectiveperformance together with safe biocompatibility andappropriate tissue interactions. The lack of well-structuredprospective multicenter clinical trials hinders theachievement of conclusive data on the efficacy andcomparative performance of anti-infective biomaterials [16].

Acknowledgments:The contributors express their gratitude for the funding by the project by Shumen University project RD 08-266/

10.03.2015 Department of Biology, RD 08-248/06.03.2015 Department of Organic Chemistry & Technology and HumanResources Development Operational Programme of the European Union (BG051PO001-3.3.06-0003/2012).

796 http://www.journal-imab-bg.org / J of IMAB. 2015, vol. 21, issue 2/

1. Gottenbos B. The developmentof antimicrobial biomaterial surfaces.Groningen: s.n., 2001. 120 p.

2. Gristina AG. Biomaterial-centered infection: microbial adhesionversus tissue integration. Science. 1987Sep 25;237(4822):1588-95. [PubMed][CrossRef]

3. Dankert J, Hogt AH, Feijen J.Biomedical polymers: Bacterial adhe-sion, colonization and infection. CRCCritical Reviews in Biocompatibility.1986; 2:219-301.

4. Denstedt JD, Wollin TA, Reid G.Biomaterials used in urology: currentissues of biocompatibility, infection,and encrustation. J Endourol. 1998Dec;12(6):493-500. [PubMed]

5. Costerton JW, Stewart PS,Greenberg EP. Bacterial biofilms: acommon cause of persistent infections.Science. 1999 May 21;284(5418):1318-22. [PubMed]

6. Gilbert P, Das J, Foley I. Biofilmsusceptibility to antimicrobials. AdvDent Res. 1997 Apr;11(1):160-7.[PubMed] [CrossRef]

7. van Loosdrecht MC, LyklemaJ, Norde W, Zehnder AJ. Influence of

interfaces on microbial activity.Microbiol Rev. 1990 Mar;54(1):75-87.

8. Christensen GD, Baddour LM,Hasty DL, Lowrance JH, Simpson WA.Microbialand foreign body factors inthe pathogenesis of medical device in-fections. In: Bisno AL, Waldvogel FA,editors. eds. Infections associated withindwelling medical devices. Washing-ton, DC: American Society for Micro-biology; 1989: 27–59.

9. Van Oss CJ. The forces involvedin bioadhesion to flat surfaces and par-ticles – their determination and relativeroles. Biofouling. 1991; 4(1-3):25-35.[CrossRef]

10. Ignatova-Ivanova Ts, Ivanov R.Exopolysaccharides from Lactic acidBacteria as Corrosion Inhibitors. Jour-nal of Life Sciences. 2014; 8(12): 940-945.

11. Ignatova-Ivanova Ts, Ivanov R.Study of Biofilm formed by lactic acidbacteria on the surface of mild steel.Journal of Life Sciences. 2014; 8(10):799-804.

12. Ignatova-Ivanova Ts, Ivanov R.Anticorrosion Effect of Biofilm Form-ing by Lactobacillus Strains on Metal

Address for correspondence:Tsveteslava V. Ignatova-IvanovaDepartment of Biology Faculty of Natural Sciences, University of Shumen,115, Universitetska Str., 9712 Shumen, Bulgaria.Phone: 0887 160 771E-mail: radi_cvet@abv.bg

Surfaces. Bulgarian Journal of Agri-cultural Science. 2013; 19(2):83-85.

13. Ignatova-Ivanova Ts, Ibrjam S.Ivanov R. Study of the Effect of Lac-tic Acid Fermentation End Products onthe Speed of the Corrosion Process. IntJ Curr Microbiol App Sci . 2015;4(4):397-401.

14. Page K, Wilson M, Mordan NJ, Chrzanowski W, Knowles J, Parkin IP.Study of the adhesion of Staphylococ-cus aureus to coated glass substrates.Journal of Materials Science. 2011Oct;46 (19): 6355-6363. [CrossRef]

15. Arciola CR, Campoccia D,Speziale P, Montanaro L, CostertonJW. Biofilm formation in Staphyloco-ccus implant infections. A review ofmolecular mechanisms and implica-tions for biofilm-resistant materials.Biomaterials. 2012 Sep;33(26):5967-5982. [PubMed] [CrossRef]

16. Campoccia D, Montanaro L,Arciola CR. A review of the biomate-rials technologies for infection-resist-ant surfaces. Biomaterials. 2013Nov;34(34):8533-8554. [PubMed][CrossRef]

REFERENCES:

Please cite this article as: Ignatova-Ivanova TV, Ibrjam SF, Ismailov IE, Christov VC, Ivanov RI. Adhesion andsurface growth of Staphylococcus aureus and Lactobacillus plantarum on various metals. J of IMAB. 2015 Apr-Jun;21(2):793-796. DOI: http://dx.doi.org/10.5272/jimab.2015212.793

Received: 24/03/2015; Published online: 14/06/2015