Extracellular Polymeric Substances of Listeria Monocytogenes

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 &amp Wingender, 2010). Bacteria release EPS whensubjected to significant physiologic stress. Bacteria embed itself inEPS during the process of biofilm formation (Marvasi,Visscher &amp 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 &amp 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&amp 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 &amp 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 &amp 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 &amp 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.

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