ExtracellularPolymeric Substances of ListeriaMonocytogenes
Results
Theextracellular polymeric substances (EPS) were extracted from theListeriamonocytogenes planktoniccells after 0, 1, 3, 6, 7, 8, 24, and 30 hours. The protein contentin the extracellular polymeric substances of L.monocytogenes wasmeasured by Bradford assay. Refer to Figure 1. The protein content inthe EPS appeared to increase with time with the average rate of0.0131mg/ml per hour (from the equation y = 0.0131x + 1.0606). The R2valueof 0.886 illustrates that the line of best fit significantlyrepresented the plot points since the value was approaching 1.
Takingthe initial protein concentration, at time 0 as 1.0338mg/ml as thereference point (Table 1), the rate of protein synthesis after 6hours was ((1.1719-1.0338mg/ml)/6hrs) 2.3017×10-2mg/mlper hour and ((1.4198-1.0338mg/ml)/24hrs) 1.608×10-2mg/mlper hour after 24 hours. Therefore, the rate of synthesis of EPSprotein content seemed to have diminished with time. Refer to Table 1and Figure 1.
Table1: Concentration of EPS Protein Content with Time (Data used forgeneration of Figure 1)
Time(h) |
Con(mg/ml) |
Stdev |
0 |
1.033829 |
0.034583 |
1 |
1.119105 |
0.01376 |
3 |
1.138214 |
0.00341 |
6 |
1.171942 |
0.017583 |
7 |
1.048121 |
0.020335 |
8 |
1.155243 |
0.067715 |
24 |
1.419807 |
0.028011 |
30 |
1.431236 |
6.15E-05 |
Figure1: The Protein Content in EPS of L.monocytogenes
Table2: Concentration of EPS Polysaccharide Content with Time (Data usedfor generation of Figure 2)
Time |
Con (mg/ml) |
Stdev |
0 |
0.094616 |
0.000205 |
1 |
0.10273 |
0.000478 |
3 |
0.107608 |
0.005464 |
6 |
0.113452 |
0.007991 |
7 |
0.125912 |
0.001844 |
8 |
0.117895 |
0.007718 |
24 |
0.150013 |
0.000137 |
30 |
0.156678 |
0.005191 |
Takingthe initial polysaccharide concentration, at time 0 as 0.09462 mg/mlas the reference point (Table 2), the rate of polysaccharidesynthesis after 6 hours was ((0.1135-0.0962mg/ml)/6hrs) 2.883×10-3mg/mlper hour and was ((0.1500-0.09462mg/ml)/24hrs) 2.308×10-3mg/mlper hour after 24 hours. Therefore, the rate of synthesis of EPSpolysaccharide content seemed to have diminished with time.
Figure2: The Polysaccharide Content in EPS of L.monocytogenes
TheEPS extracted from L.monocytogenes planktoniccells after 0, 1, 3, 6, 7, 8, 24 and 30 hours. The polysaccharidecontent in EPS, of the L.monocytogenes wasdetermined using the phenol-sulfuric acid method. Refer to Figure 2.The polysaccharide content in EPS appeared to increase time with anaverage rate of 0.0019 mg/ml per hour. The rate was derived from theequation (y = 0.0019x + 0.1019) in Figure 2 above. The R2valueof 0.948 illustrate that the line of best fit significantlyrepresented the plot points since the value was approaching 1.
Therate of increase of protein content (0.0131mg/ml per hour) surpassesthat of carbohydrates (0.0019 mg/ml per hour). Despite the variationof protein and polysaccharide content in the secreted EPS the studydemonstrated that the two substances form parts of the L.monocytogenes biofilm.Biofilms are microbial colonies that establish themselves indifferent abiotic and biotic services (Dusane, etal.,2013). Biofilm formation is a shared characteristic among diversebacteria species whereby majority of them are pathogenic (Denef, etal.,2010). Besides, it showed that the concentrations of polysaccharidesand protein in the EPS of tend to increase with time. The findingsdemonstrate that the biofilm thickness may be increasing with timeand the L.monocytogenes cellsdivides and multiply.
Discussion
Thefindings demonstrated that polysaccharides and proteins arecomponents of the EPS however, rates of synthesis differ. Otherconstituents of the EPS include nucleic acids, and lipids. Proteinsand polysaccharides contribute the mechanical stability of thebiofilms. EPS is itself an important constituent of the microbialbiofilms (Flemming & Wingender, 2010). Bacteria release EPS whensubjected to significant physiologic stress. Bacteria embed itself inEPS during the process of biofilm formation (Marvasi,Visscher & Martinez, 2010).
Bacteriaembed itself in EPS as a protection strategy in response of adverseenvironmental conditions and facilitate infection process(Sharma, etal.,2016). Biofilms are associated with infectious bacterial pathogens(Boucher, etal.,2009). Physiological stress activates cyclic di-GMP, which is asignaling molecule to produce bacterial cellulose. Bacterialcellulose is an important component of the microbial biofilm (Morgan,McNamara & Zimmer, 2014). EPS is the primary functional andstructural constituents of microbial biofilms (Jachlewski, etal., 2015). Since proteins and polysaccharides are the major constituent of theEPS, they are also important elements that support microbial biofilmprocess (Jeans, etal., 2008Jiao, etal., 2011).
Inthis experiment the rate of protein synthesis (0.0131mg/ml per hour)is greater than that of polysaccharides (0.0019 mg/ml per hour)synthesis. The protein requirement in the EPS is greater than that ofpolysaccharides however, the rates of synthesis for both diminishedwith time. L.monocytogenes synthesizesa biofilm which it shields it from dehydration (Flemming& Wingender, 2010).
Theproteins detected on the L.monocytogenes mayhave cytoplasmic origin. Majority of the proteins are released viathe cell membrane vesicles and the biofilm-intrinsic cell lysis inthe course of biofilm maturation (Jachlewski, etal.,2015). The observed protein content in this study may have similarorigins. The cationic polysaccharide adhesins generates a matrix thatlinks cells together during biofilm formation. Polysaccharidesstrengthen cell-to-cell attachment thus facilitating for microbialaggregation and biofilm synthesis (Formosa-Dague, etal., 2016).
TheEPS are the metabolic products that accumulate on the cell surface ofbacteria. The EPS generates a protective layer over the surfaces(Wilmes, etal., 2009).The acidogenic sludge is characterized by predominant carbohydratecontent whereas the methanogenic sludge is characterized bypredominant protein content (Chen, etal., 2014Liu & Fang, 2002). Different extracellular matrix quantificationstrategies are available. In this study, the Bradford Assay wasemployed for the determination of protein content whereas, thephenol-sulfuric method was used for the determination of carbohydratecontent in the EPS matrix of L.monocytogenes. Thebiofilms are generally produced under optimal conditions in alaboratory environment (Combrouse,etal., 2013).Themicrogram quantities of protein in a substance can be determinedthrough the utilization of Bradford Comassie brilliant, followed by aphotometric detection at 590nm. The dye enables easy and efficientquantification of protein in cell lysates, recombinant proteinsamples, and cellular fractions (Ernst & Zor, 2010 Tiensuu, etal., 2013).
Thebiofilm-secreting bacterial pathogens are typically difficult tomanage or treat with conventional anti-bacterial agents(Cywes-Bentley,etal., 2013Lourenco, etal.,2013). The biofilm formation capability makes L.monocytogenes animportant in health and food industry since it imparts resistance todetergent and antibacterials (Aguilera, etal.,2008). Biofilm-forming bacteria are commonly found on medical devices(Revdiwala, Rajdev & Mulla, 2012). Besides microbial biofilms canblock pipes and confined fluid channels (Lear, etal., 2009).EPS is composed of extracellular DNA, polysaccharides and proteinsand other substances such as extracellular matrix (Colagiorgi, etal., 2016Scallan,etal., 2011).Teichoic acid is a vital component of the polysaccharide content in agiven bacterial biofilm (Brauge, etal., 2016).The biofilm-producing bacteria as often linked with hospital acquiredinfections including infections caused by L.monocytogenes, Staphylococcus aureus andStaphylococcusepidermidis (Gutiérrez,etal., 2015).
Themicrobial biofilms consist of bacterial cells with different activitystates. Proteins and polysaccharides are synthesized at differentrates. Different constituents of the EPS are coded and regulated bydifferent genes (Lo, etal., 2007Rani, etal., 2007).The findings of the majority of the literature sources are consistentwith the findings of this study. In this study, significantconcentrations of proteins and polysaccharides were detected as themajor components of the L.monocytogenes secretedEPS.
Theexopolysaccharides are the primary elements of the extracellularmatrix in various microorganisms. The L.monocytogenes strainshave been characterised by the formation of the carbohydratecontaining extracellular matrix. A total of 30 gene targets areresponsible for the generation of L.monocytogenes biofilms.It includes the phosphate-sensing two-component system referred to asphoPRandthe D-alanylation pathway referred to as dltABCD.Deletionof phoPRanddltABCDresultsin a significant reduction of the capacity to produce biofilms. Thephosphate-sensing phoPRtwo-componentsystem and D-alanylation of lipoteichoic acids are two importantplayers that support the formation of biofilms. Besides, the proteincontent of the L.monocytogenes isdemonstrated by the dispersal of bacterial cells or disintegration ofbiofilms when treated with proteases (Colagiorgi, etal., 2016).
Theliterature review demonstrated that no studies have been carried outto study the rate of synthesis of proteins and polysaccharides in themicrobial biofilms. Therefore, this is a pioneer study in comparingthe rate of polysaccharide and protein synthesis as the primarycomponents of the microbial biofilms. Both polysaccharides andproteins are the major adhesion factors in the microbial biofilmsthat facilitate for cell to cell attachment and aggregation ofbacterial cells and finally generate a significant biofilm layer. Thefindings showed that the rate of protein synthesis is greater thanpolysaccharide synthesis however, the rates of production of the twoconstituents diminished with time. It implies that the rate ofprotein and polysaccharide synthesis in L.monocytogenesis at its peak in the absence of biofilms but reduces as the biofilmis generated. The investigator recommends further studies to betterunderstand and explore the area in the future.
References
Aguilera,A., Souza-Egipsy, V., Martin-Uriz, P.S., & Amils, R. (2008).Extracellular matrix assembly in extreme acidic eukaryotic biofilmsand their possible implications in heavy metal adsorption. Aquat.Toxicol.,88,257-266.
Boucher,H.W., Talbot, G.H., Bradley, J.S., Edwards, J.E., Gilbert, D., etal.(2009). Bad bugs, no drugs: no ESKAPE! An update from the InfectiousDiseases Society of America. ClinInfect Dis,48,1–12.
Brauge,T., Sadovskaya, I., Faille, C., Benezech, T., Maes, E., Guerardel,Y., & Midelet-Bourdin, G. (2016). Teichoic acid is the majorpolysaccharide present in the Listeriamonocytogenes biofilmmatrix. FEMSMicrobiol. Lett., 363. doi:10.1093/femsle/fnv229.
CDC.(2014). Incidence and trends of infection with pathogens transmittedcommonly through food-foodborne diseases active surveillance network,10 US sites, 2006–2013. Morb.Mortal. Wkly. Rep. 63,328–332.
Chen,L.H., Köseoglu, V.K., Guvener, Z.T., Myers-Morales, T., Reed, J.M.,D’Orazio, S.E., Miller, K.W., & Gomelsky, M. (2014). Cyclicdi-GMP-dependent signaling pathways in the pathogenicfirmicute Listeriamonocytogenes. PLoSPathog.10. doi:10.1371/journal.ppat.1004301. 
Colagiorgi,A., Ciccio, P., Zanardi, E., Ghidini, S., & Lanieri, A. (2016). Alook inside the Listeriamonocytogenes BiofilmsExtracellular Matrix. Microorganisms,4(3),22. doi: 10.3390/microorganisms4030022.
Combrouse,T., Sadovskaya, I., Faille, C., Kol, O., Guérardel, Y., &Midelet-Bourdin, G. (2013). Quantification of the extracellularmatrix of the Listeriamonocytogenes biofilmsof different phylogenic lineages with optimization of cultureconditions. J.Appl. Microbiol. 114,1120–1131. doi: 10.1111/jam.12127.
Cywes-Bentley,C., Skurnik, D., Zaidi, T., Roux, D., DeOliveira, R.B., Garrett,W.S., Lu, X., O’Malley, J., Kinzel, K., & Zaidi, T., etal.(2013). Antibody to a conserved antigenic target is protectiveagainst diverse prokaryotic and eukaryotic pathogens. Proc.Natl. Acad. Sci. USA.110,E2209–E2218. doi: 10.1073/pnas.1303573110.
Denef,V.J., Kalnejais, L.H., Mueller, R.S., Wilmes, P., Baker, B.J.,Thomas, B.C., VerBerkmoes, N.C., Hettich, R.L., & Banfield. J.F.(2010). Proteogenomic basis for ecological divergence of closelyrelated bacteria in natural acidophilic microbial communities. Proc.Natl. Acad. Sci. U. S. A.,107,2383-2390.
Dusane,D.H., Damare, S.R., Nancharaiah, Y.V., Ramaiah, N., Venugopalan,V.P., Kumar, A.R., & Zinjarde1, S.S. (2013). Disruption ofMicrobial Biofilms by an Extracellular Protein Isolated fromEpibiotic Tropical Marine Strain of Bacilluslicheniformis. PLoS One, 8(5),e64501. doi: 10.1371/journal.pone.0064501.
EFSA.(2015). The European Union summary report on trends and sources ofzoonoses, zoonotic agents and food-borne outbreaks in 2013. EFSAJ.13,165.
Ernst,O., & Zor, T. (2010). Linearization of the Bradford ProteinAssay. JoVE,30. Retrievedon Jan 12, 2017 from, http://www.jove.com/details.php?id=1918.
Flemming,H-C., & Wingender, J. (2010). The biofilm matrix. NatureReviews Microbiology, 8,623-633. Doi:10.1038/nrmicro2415
Formosa-Dague,C., Feuillie, C., Beaussart, A., Derclaye, S., Kucharíková, S.,Lasa∥,I., Dijck, P., & Yves F. Dufrêne, Y.F. (2016). Sticky matrix:Adhesion mechanism of the staphylococcal polysaccharidesintercellular adhesin. ACSNano, 10(3),3443-3452. DOI: 10.1021/acsnano.5b07515.
Gutiérrez,D., Briers, Y., Rodríguez-Rubio, L., Martínez, B., Rodríguez, A.,Lavigne, R., & García, P. (2015). Roleof the Pre-neck Appendage Protein (Dpo7) from Phage vB_SepiS-phiIPLA7as an Anti-biofilm Agent in Staphylococcal Species. Frontiersin Microbiology, 6(1315),1-10. doi: 10.3389/fmicb.2015.01315
Jachlewski,S., Jachlewski, W.D., Linne, U., Bräsen, C., Wingender, J., &Siebers1,B. (2015). Isolation of extracellular polymeric substancesfrom biofilms of the thermoacidophilic archaeon Sulfolobusacidocaldarius. Front Bioeng Biotechnol.,3,123.doi: 10.3389/fbioe.2015.00123
Jeans,C., Singer, S.W., Chan, C.S., Verberkmoes, N.C., Shah, M., Hettich,R.L., Banfield, J.F., & Thelen, M.P. (2008).Cytochrome 572 is a conspicuous membrane protein with iron oxidationactivity purified directly from a natural acidophilic microbialcommunity. ISMEJ. 2,542-550
Jiao,Y., D`haeseleer, P., Dill, B.D., Shah, M., VerBerkmoes, N.C.,Hettich, R.L., Banfield, J.F., & Thelen, M.P. (2011).Identification of biofilm matrix-associated proteins from an acidmine drainage microbial community. Appl.Environ. Microbiol., 77(15),5230-5237. Doi: 10.1128/AEM.03005-10
Lear,G., Niyogi, D., Harding, J., Dong, Y., & Lewis, G. (2009).Biofilm bacterial community structure in streams affected by acidmine drainage. Appl.Environ. Microbiol.,75,3455-3460.
Liu,H., & Fang, H.P. (2002). Extraction of extracellular polymericsubstances (EPS) of sludges. Journalof Biotechnology, 95,249-256.
Lo,I., Denef, V.J., Verberkmoes, N.C., Shah, Goltsman, D., DiBartolo,G., Tyson, G.W., Allen, E.E., Ram, R.J., Detter, J.C., Richardson,P., Thelen, M.P., Hettich, R.L., & Banfield, J.F. (2007).Strain-resolved community proteomics reveals recombining genomes ofacidophilic bacteria.Nature,446,537-541.
Lourenco,A., de Las Heras, A., Scortti, M., Vazquez-Boland, J., Frank, J.F., &Brito, L. (2013). Comparison of Listeriamonocytogenes exoproteomesfrom biofilm and planktonic state: Lmo2504, a protein associated withbiofilms. Appl.Environ. Microbiol. 79,6075–6082. doi: 10.1128/AEM.01592-13.
Marvasi,M., Visscher, P.T., & Martinez, L.C. (2010). Exopolymericsubstances (EPS) from Bacillussubtilis: polymersand genes encoding their synthesis. FEMSMicrobiology Letters, 313(1),1-9. DOI: 10.1111/j.1574-6968.2010.02085.x.
Morgan,J.L., McNamara, J.T., & Zimmer, J. (2014). Mechanism ofactivation of bacterial cellulose synthase by cyclic di-GMP. NatureStructural & Molecular Biology, 21,489-496. Doi:10.1038/nsmb.2803.
Rani,S.A., Pitts, B., Beyenal, H., Veluchamy, R.A., Lewandowski, Z.,Davison, W.M., Buckingham-Meyer, K., & Stewart, P.S. (2007).Spatial Patterns of DNA Replication, Protein Synthesis, and OxygenConcentration within Bacterial Biofilms Reveal Diverse PhysiologicalStates.Journalof Bacteriology, 169(11),4223-4233.
Revdiwala,S., Rajdev, B.M., & Mulla, S. (2012). Characterization ofbacterial etiologic agents of biofilm formation in medical devices incritical care setup. CritCare Res Pract., 2012,945805.
Scallan,E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M.A., Roy,S.L., Jones, J.L., & Griffin, P.M. (2011). Foodborne illnessacquired in the united states-major pathogens. Emerg.Infect. Dis. 17,7–15. doi: 10.3201/eid1701.P11101
Sharma,G., Sharma, S., Sharma, P., Chandola, D., Dang, S., Gupta, S., &Gabrani, R. (2016). Escherichiacoli biofilm:development and therapeutic strategies. Journalof Applied Microbiology, 121(2),309-319. DOI: 10.1111/jam.13078.
Tiensuu,T., Andersson, C., Rydén, P., & Johansson, J. (2013). Cycles oflight and dark co-ordinate reversible colony differentiationin Listeriamonocytogenes. Mol.Microbiol.,87,909–924. doi: 10.1111/mmi.12140.
Wilmes,P., Remis, J.P., Hwang, M., Auer, M., Thelen, M.P. & Banfield,J.F. (2009). Natural acidophilic biofilm communities reflect distinctorganismal and functional organization.ISME J.3,266-270.