Quantification Of Exopolysaccharides Research Articles

Bio-Prospecting Xylose-Utilizing, Exopolysaccharide (EPS)-Producing Bacteria and EPS Quantification through Submerged Fermentation using Xylose as the Major Carbon Source

Study’s Novelty/ Excerpt This study identified bacterial isolates from diverse environments capable of utilizing xylose for exo-polysaccharide (EPS) production, an area with limited prior research. Potential EPS producers were isolated and identified as Staphylococcus, Enterobacter, Kocuria, Klebsiella, Enterococcus, Serratia, and Burkholderia species using a salt-based medium with xylose as the primary carbon source. The highest EPS production was observed in Enterobacter cloacae D1, E. cloacae D2, Klebsiella oxytoca D2, and K. oxytoca G1, ranging from 0.04 g/L to 2.0 g/L, highlighting these isolates' potential for optimization and application in various industries. Full Abstract Many microorganisms are capable of producing Exo-polysaccharides (EPS) while utilizing simple sugars and hexoses. These EPS found applications in various fields, such as agricultural biotechnology, pharmaceuticals, textiles, and food industries. However, there is a lack of studies on EPS-elaborating bacteria that can utilize pentoses like xylose. Therefore, the utilization of alternative carbon sources for EPS production has become a focus of recent research. This study aimed to prospect bacteria that can utilize xylose for EPS production. Samples from agricultural soil, dump sites, saline soil, cement-contaminated soil, fresh cow milk, cow dung, and yogurt were serially diluted and cultured in a salt-based medium with xylose as the primary carbon source. Slimy and mucoid colonies were selected as potential EPS-producing isolates and identified morphologically and biochemically using the VITEK 2 Automated identification system. The quantification of EPS production by these isolates was conducted through submerged fermentation with xylose as the sole carbon source. The mean heterotrophic bacterial count of xylose-utilizing bacteria ranged from 2.1x106 CFU to 3.5x108 CFU per gram of the samples analyzed. The slimy and mucoid colonies were identified as members of the genera Staphylococcus, Enterobacter, Kocuria, Klebsiella, Enterococcus, Serratia, and Burkholderia. The quantities of EPS produced by the isolates ranged from 0.04 g/L to 2.0 g/L, with E. cloacae D1, E. cloacae D2, K. oxytoca D2, and K. oxytoca G1 elaborating the highest amount of EPS. Bacterial isolates capable of utilizing xylose for EPS production were obtained from various sources, showing potential for further optimization

Anti-quorum sensing activity of selected cationic amino acids against Chromobacterium violaceum and Pseudomonas aeruginosa

Antibiotic-resistant strains and "superbugs" have emerged as a result of unrestricted use and overuse of antibiotics. N-Acyl homoserine lactones, also known as autoinducers, are unique type of signaling molecule and passively permeate to the cells of Gram-negative bacteria. Accumulation of signaling molecules in the environment regulates the bacterial population. The organisms Pseudomonas aeruginosa and Chromobacterium violaceum are Gram-negative bacteria when exposed to humans, cause fatal infections. The selected cationic amino acids histidine, arginine, and ornithine were assessed for their anti-quorum sensing activity against the P. aeruginosa and C. violaceum. The MIC and sub-MIC concentrations of the selected amino acids have been found by the broth dilution method. Both the cultures have been treated with MIC and sub-MIC levels and the virulence factors like rhamnolipid, protease, elastase, pyoverdine, pyocyanin for P. aeruginosa and violacein, chitinase for C. violaceum is quantified. The quantification of exo-polysaccharides and the biofilm as well as visualization by confocal laser scanning microscope have been done for both organisms. The movement of the cells was assessed by swarming and swimming motility in both organisms. The genes activated for all these virulence factors were quantified by RT-qPCR and expressed in fold change. From the results, it is evident that the histidine inhibited both the organisms at concentrations of MIC and sub-MIC more than the percentage of virulence factors production by arginine and ornithine. The selected cationic amino acids prove to be an effective compound in antimicrobial therapeutics in order to reduce opportunistic bacterial infections.

Bradyrhizobium diazoefficiens USDA 110 displays plasticity in the attachment phenotype when grown in different soybean root exudate compounds.

Bradyrhizobium diazoefficiens, a symbiotic nitrogen fixer for soybean, forms nodules after developing a symbiotic association with the root. For this association, bacteria need to move toward and attach to the root. These steps are mediated by the surface and phenotypic cell properties of bacteria and secreted root exudate compounds. Immense work has been carried out on nodule formation and nitrogen fixation, but little is known about the phenotype of these microorganisms under the influence of different root exudate chemical compounds (RECCs) or how this phenotype impacts the root attachment ability. To address this knowledge gap, we studied the impact of 12 different RECCs, one commonly used carbon source, and soil-extracted solubilized organic matter (SESOM) on attachment and attachment-related properties of B. diazoefficiens USDA110. We measured motility-related properties (swimming, swarming, chemotaxis, and flagellar expression), attachment-related properties (surface hydrophobicity, biofilm formation, and attachment to cellulose and soybean roots), and surface polysaccharide properties (colony morphology, exopolysaccharide quantification, lectin binding profile, and lipopolysaccharide profiling). We found that USDA 110 displays a high degree of surface phenotypic plasticity when grown on the various individual RECCs. Some of the RECCs played specific roles in modulating the motility and root attachment processes. Serine increased cell surface hydrophobicity and root and cellulose attachment, with no EPS formed. Gluconate and lactate increased EPS production and biofilm formation, while decreasing hydrophobicity and root attachment, and raffinose and gentisate promoted motility and chemotaxis. The results also indicated that the biofilm formation trait on hydrophilic surfaces (polystyrene) cannot be related to the attachment ability of Bradyrhizobium to the soybean root. Among the tested phenotypic properties, bacterial cell surface hydrophobicity was the one with a significant impact on root attachment ability. We conclude that USDA 110 displays surface plasticity properties and attachment phenotype determined by individual RECCs from the soybean. Conclusions made based on its behavior in standard carbon sources, such as arabinose or mannitol, do not hold for its behavior in soil.

Guaiacol augments quorum quenching potential of ciprofloxacin against Pseudomonas aeruginosa.

The present study aims to investigate the antimicrobial as well as antivirulence potential and the principle mechanism of action of guaiacol against Pseudomonas aeruginosa. Quorum sensing inhibition and membrane disruption studies were performed to check the effect of guaiacol on the virulence of P. aeruginosa. Production of various virulence factors and biofilm formation was studied at a sub-MIC concentration of guaiacol alone (1/8 MIC) and in combination with ciprofloxacin (1/2 FIC). Guaiacol exhibited synergistic interactions with ciprofloxacin and further reduced the production of all virulence factors and biofilm formation. Using crystal violet (CV) assay and quantification of exopolysaccharide, we observed weak biofilm formation, together with reduced motilities at sub-MIC, which was further visualized by confocal laser microscopy and Field Emission Scanning Electron Microscopy. The antibacterial activity of guaiacol against P. aeruginosa upon 2 × MIC exposure coincided with enhanced membrane permeability leading to disruption and release of cellular material as quantified by CV uptake assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results demonstrated that sub-MICs of guaiacol in combination with ciprofloxacin can act as a potent alternate compound for attenuation of quorum sensing in P. aeruginosa. The study reports that guaiacol in combination with ciprofloxacin at 1/2 FIC significantly compromised the bacterial growth and motilities alongside inducing quorum quenching potential. This was accompanied by inhibition of biofilm which subsequently decreased EPS production at sub-MIC concentration. Furthermore, guaiacol in combination displayed a severe detrimental effect on bacterial membrane disruption, thereby enhancing cellular material release. For the first time, the potential of guaiacol in combination with ciprofloxacin in attenuation of virulence factors, and biofilm formation in Pseudomonas aeruginosa was described. Results corroborate how plant bioactive in synergism with antibiotics can act as an alternate treatment regime to tackle the menace of drug resistance.

Diversity of Ralstonia pseudosolanacearum, the causal agent of bacterial wilt on Eucalyptus pellita in Indonesia

Abstract. Siregar BA, Giyanto, Hidayat SH, Siregar IZ, Tjahjono B. 2021. Diversity of Ralstonia pseudosolanacearum, the causal agent of bacterial wilt on Eucalyptus pellita in Indonesia. Biodiversitas 22: 2538-2545. The Ralstonia species complex was initially classified into five races and five biovars but the classification could not accommodate the isolates' phylogenetic history or geographic origins. A phylotype and sequevar system is based on the geographic distribution and characteristics of endoglucanase (egl) and hypersensitive response and pathogenicity (hrp) gene sequences. This study aims to describe pathogen diversity of the causal agent of bacterial wilt on Eucalyptus pellita F. Muell. Pathogens were isolated from wilting seedlings and trees in several Eucalyptus plantations. The phenotypic diversity analysis included biovar, exopolysaccharide quantification and virulence tests, while genotypic diversity included phylotypes and sequevar determination based on egl gene sequences. A total of 35 strains were isolated from the field and nurseries of Eucalyptus in various locations. All isolates were confirmed as Ralstonia species complex based on morphological characteristics and molecular studies using species-specific primers. These isolates were dominantly classified as biovar 3 and 4 and had a high variation on virulence and EPS production. Based on the egl sequence's alignment, 29 strains of Phylotype I are grouped into four sequevar references (sequevars 14, 17, 18, 30) and new a sequevar 58. This study shows that strains of R. pseudosolanacearum causing bacterial wilt on E. pellita in Indonesia have high phenotypic and genotypic diversities.

Preparative HPLC fraction of Hibiscus rosa-sinensis essential oil against biofilm forming Klebsiella pneumoniae.

Recent years Klebsiella pneumoniae (K. pneumoniae) biofilm formation (BF) is emerging thread worldwide. For tackling this problem, we have chosen Hibiscus rosa-. pneumoniae. The HPLC purified essential oils (EOs sinensis (H. rosa-sinensis) (HRS) to inhibit the BF K) of H. rosa-sinensis was performed against BF K. pneumoniae and showed concentration dependent biofilm inhibition. At the MBIC of EOs (90 µg/ml), the biofilm inhibition was showed at 92% against selected BF K. Pneumoniae. The biofilm metabolic assay, exopolysaccharide quantification and hydrophobicity index variation results exhibited with 88%, 92% and 89% reduction at 90 μg/mL was observed respectively. In addition, the morphological modification of MBIC treated K. pneumoniae was clearly viewed by scanning electron microscope (SEM). Overall, all the invitro experiments result were confirmed that the MBIC of H. rosa-sinensis EOs was very effective against BF K. pneumonia.

Efficiency of purification methods on the recovery of exopolysaccharides from fermentation media

De-Man Rogosa and Sharpe (MRS) is a complex medium commonly used to obtain exopolysaccharides (EPS) from lactic acid bacteria. However, the various nutrients (carbon and nitrogen sources) of media and supplements added to enhance the bacterial growth and EPS production, may interfere with the purification of the extracts resulting in an over-estimation of the EPS and erroneous structural assignments. The efficiency of trichloroacetic acid (TCA)/pronase and 5-sulfosalicylic acid – SSA methods was evaluated. In addition, the interference of the major MRS broth components as well as lactose was evaluated using xanthan gum as model control EPS. It was concluded that MRS medium is a major source of interfering compounds in the quantification of EPS, mainly glucose-rich material and to a lesser extent mannoproteins from yeast extract. This effect was found to be potentiated by the presence of lactose. TCA/pronase method was shown to be more efficient in eliminating interferents.

Antibacterial synergy between rutin and florfenicol enhances therapeutic spectrum against drug resistant Aeromonas hydrophila

Emergence of antibiotic resistant bacteria has necessitated the drive to explore competent antimicrobial agents or to develop novel formulations to treat infections including Aeromonas hydrophila. The present study investigates the synergistic antibacterial effects of citrus flavonoid rutin and florfenicol (FF) against A. hydrophila in vitro and in vivo. Rutin is extracted and purified from Citrus sinensis peel through preparative HPLC and characterized through TLC, GC-MS and 1H and 13C NMR analyses. Though rutin did not display significant antibacterial activity, it modulated FF activity resulting in four-fold reduction in the MIC value for FF. The anti-biofilm potential of synergistic association of rutin and FF was validated by protein analysis, quantification of exopolysaccharide (EPS) and microscopy studies using sub-MIC doses. Besides antibacterial action, in vivo studies showed that Rutin/FF combination enhanced host immunity by improving blood cell count, anti-protease, and lysozyme activities as well as decreased the oxidative stress and the pathological changes of tilapia Oreochromis niloticus against A. hydrophila infection. No significant DNA damages or clastogenic effects were detected in tilapia challenged with A. hydrophila under Rutin/FF treatment. It is shown that an acute-phase Lipopolysaccharide binding protein (LBP) enhances the innate host defence against bacterial challenge. Semi quantitative RT-PCR and western blot results revealed the significant increase of LBP in the supernatant of tilapia monocytes/macrophages challenged with A. hydrophila upon treatment. The study findings substantiate that the combination of natural molecules with antibiotics may open up possibilities to treat MDR strains.

Combined effect of a natural flavonoid rutin from Citrus sinensis and conventional antibiotic gentamicin on Pseudomonas aeruginosa biofilm formation

Extensive and irrational use of antibiotics to treat biofilm associated infections has led to the emergence of multiple drug resistant (MDR) strains. Rutin, a vitamin P rich flavonoid group of phytochemical with antimicrobial property, has been studied for its anti-biofilm potential against Pseudomonas aeruginosa, which is a model biofilm forming pathogenic bacterium, in combination with conventional antibiotic gentamicin. For this study, rutin was isolated from the peels of Citrus sinensis by column chromatography and HPLC and characterized by UV-vis and FT-IR spectral analyses. Results showed that rutin exhibited minimum inhibitory concentration (MIC) at 800 μg/ml against the bacterium. Its anti-biofilm potential was examined by crystal violet staining, protein analysis, microscopy studies and quantification of exopolysaccharide (EPS) using several sub-MIC doses. The inhibition of biofilm formation was synergistically enhanced when the bacterium was treated with rutin (200 μg/ml) along with an antibiotic gentamicin (2.5 μg/ml). EPS production was responsible for adherence and maturation of biofilms, which was also significantly reduced in the tested strain. To the best of our knowledge, this is the first study describing the anti-biofilm potential of citrus flavonoid rutin as well as its synergistic effects when combined with gentamicin against P. aeruginosa. Findings of the study substantiate that rutin can be potentially used as an adhesion inhibitor for the treatment of P. aeruginosa infections.

Biofilm formation and exopolysaccharide (EPS) production by Cronobacter sakazakii depending on environmental conditions

Biofilm matrices are formed largely of extracellular polymeric substance (EPS). This study was conducted to investigate biofilm formation and EPS production by Cronobacter sakazakii under various conditions (media, nutrition, and relative humidity (RH)) by quantification of EPS and cell populations, Field Emission Scanning Electron Microscope (FE-SEM), and colony observation. Various agar media conditions (TSA without dextrose (W/D), M9 minimum salt medium (MSM) agar, and M9 MSM agar with 3% glucose, 3% NaCl, 3% Tween 80, 3% sucrose, and adjusted to pH 5 with HCl) were prepared. C. sakazakii biofilm formed on the surface of stainless steel coupons (SSCs) immersed in TSB W/D and M9 MSM with or without 0, 1, 3, and 5% sucrose and subsequently exposed to various RH levels (23, 43, 68, 85, and 100%). EPS production by C. sakazakii on TSA W/D was significantly higher than that on other media after 1 and 2 days. However, C. sakazakii ATCC 12868 produced the highest levels of EPS (209.18 ± 16.13 and 207.22 ± 4.14 μg/mL after 1 and 2 days, respectively) on M9 MSM agar with 3% sucrose. Regarding C. sakazakii ATCC 12868 biofilm formed on the surface of SSCs immersed in M9 MSM with 0, 1, 3, and 5% sucrose and subsequently exposed to various RHs, populations were significantly different among the various RHs and sucrose concentrations, and EPS production was significantly higher (4.69 mg/L) compared to other sucrose concentrations (0%:0.71 mg/L and 1%:0.98 mg/L), except for M9 MSM with 3% sucrose (2.97 mg/L) (P ≤ 0.05). From these results, biofilm formation and EPS production by C. sakazakii differed depending on the nutrient or environmental conditions provided to the cells.

New protocol for the rapid quantification of exopolysaccharides in continuous culture systems of acidophilic bioleaching bacteria

In this study, we investigate exopolysaccharide production by a bacterial consortium during the bioleaching of a cobaltiferrous pyrite. Whereas comparable studies have looked at exopolysaccharide production in batch systems, this study focuses on a continuous system comprising a series of four stirred bioreactors and reveals the difficulties in quantifying biomolecules in complex media such as bioleached samples. We also adapted the phenol/sulphuric acid method to take into account iron interference, thus establishing a new protocol for sugar quantification in bioleached samples characterised by low pH (1.4) and high iron concentration (2 g l(-1)). This allows sugar analysis without any prior sample preparation step; only a small amount of sample is needed (0.5 ml) and sample preparation is limited to a single filtration step. We found that free exopolysaccharides represented more than 80% of the total sugars in the bioreactors, probably because stirring creates abrasive conditions and detaches sugars bound to pyrite or bacteria and that they were produced mainly in the first two reactors where bioleaching activity was greatest. However, we could not establish any direct link between the measured exopolysaccharide concentration and bioleaching activity. Exopolysaccharides could have another role (protection against stress) in addition to that in bacterial attachment.

Quantification of exopolysaccharide, lactic acid, and lactose concentrations in culture broth by near-infrared spectroscopy.

Near-infrared spectroscopy (NIRS) was used for the simultaneous prediction of exopolysaccharide (EPS; 0-3 g/L) and lactic acid (0-59 g/L) productions as well as lactose (0-68 g/L) concentration in supernatant samples from pH-controlled batch cultures of Lactobacillus rhamnosus RW-9595M in supplemented whey permeate medium. To develop calibration equations, the correlation between the second derivative of 164 NIRS transmittance spectra and concentration data obtained with reference methods was calculated at the wavelength between 1653-1770 and 2041-2353 nm, using a partial least-squares method (PLS). The lactic acid and lactose concentrations were measured by HPLC, and the EPS concentration was estimated by a new ultrafiltration method. The PLS correlation coefficient (R(2)) and the standard error of cross-validation for the calibrations were 91% and 0.26 g/L for EPS, 99% and 2.54 g/L for lactic acid, and 98% and 3.32 g/L for lactose, respectively. The calibration equations were validated with 45 randomly selected culture samples from 6 cultures that were not used for calibration. A high agreement between data of the reference methods and those of NIRS was observed, with correlation coefficients and standard errors of prediction of 99% and 1.64 g/L for lactic acid, 99% and 4.5 g/L for lactose, and 91% and 0.32 g/L for EPS. The results suggest that NIRS could be a useful method for rapid monitoring and control of EPS lactic fermentations.

Development of a growth medium suitable for exopolysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus RR

Complex media are commonly used in studies examining exopolysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus. However, quantification of exopolysaccharide in complex medium can be complicated by interference due to carbohydrate polymers contained in media components. This study was undertaken to identify components of MRS, a common medium for cultivation of L. delbrueckii ssp. bulgaricus, that interfere with exopolysaccharide quantification, to develop a medium for production of exopolysaccharide that provides for growth of L. delbrueckii ssp. bulgaricus strain RR similar to MRS, and to demonstrate exopolysaccharide production by L. delbrueckii ssp. bulgaricus RR grown in the newly developed medium. Phenol-sulfuric acid determinations were conducted on uninoculated MRS broth with and without yeast extract, beef extract and proteose peptone #3. These three ingredients accounted for 94% of the total background exopolysaccharide-equivalent in MRS broth. Based on these results, a semi-defined medium (SDM) providing minimal interference was developed using yeast nitrogen base ® and Bacto casitone ®. Growth of L. delbrueckii ssp. bulgaricus RR at 42°C in semi-defined medium and MRS was evaluated, and generation times did not differ significantly (0.94 h in MRS and 0.85 h in SDM). Exopolysaccharide production by L. delbrueckii ssp. bulgaricus RR during growth in semi-defined medium was evaluated at 30 and 40°C. The rate of exopolysaccharide production was lower at 30°C (8.04 (mg/l-h) than at 40°C (11.95 (mg/l-h), but the maximal concentration of exopolysaccharide produced was similar at both temperatures.