{"id":1133,"date":"2015-03-25T09:40:46","date_gmt":"2015-03-25T09:40:46","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=1133"},"modified":"2020-04-25T10:34:27","modified_gmt":"2020-04-25T10:34:27","slug":"augmentation-of-the-growth-of-pathogenic-bacteria-by-proteins-of-tridax-procumbans-l","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol3no1\/augmentation-of-the-growth-of-pathogenic-bacteria-by-proteins-of-tridax-procumbans-l\/","title":{"rendered":"Augmentation of the Growth of Pathogenic Bacteria by Proteins of Tridax Procumbans L."},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Plant proteins are reported as the most widely used constituents in biomedical research. Pathogenic organisms often attach to human epithelial or erythrocytes cell surfaces via Plant protein-glycan interactions1<\/sup>. \u00a0Bacterial species are important opportunistic pathogens having the ability to cause diarrhoea, dysentery and extra-intestinal infections2<\/sup>. Many pathogenic bacterial species can be distinguished by their carbohydrate rich surface antigen, which is referred to as glycoepitopes. These glycoepitopes are associated with capsular polysaccharides3<\/sup>.<\/p>\n

Plant proteins are used for direct clumping of microorganisms in microbiology. Clumping of Gram-negative Diplococcus<\/em> by Wheat germ agglutinin (WGA) has been considered as positive test for N. gonorrhea<\/em>. Similarly, Bacillus anthracis<\/em> can be identified by clumping with soybean<\/em> agglutinin4<\/sup>. Plant proteins are able to selectively clump Pyogenic cocci<\/em>. Groups A, B, C, F and G streptococci can be readily distinguished by Plant proteins, however, Salmonella genus<\/em> was unable to react with Con A.5<\/sup>.<\/p>\n

The interaction between most Plant proteins and microbial surfaces are due to the glycoconjugates of microbial surfaces which is a potential site for clumping. Some Plant proteins were able to selectively aggregate bacteria6<\/sup>.<\/p>\n

Medicinal plants are routinely exploited for the study of their conventional uses through the authentication of pharmacological effects and can be natural complex sources of new anti-infectious agents. The present study aimed at evaluating the agglutination activity of plant Tridax procumbans calyx protein<\/em> against bacterial strains isolated from human infection following the agglutination assay of Tridax procumbans calyx protein<\/em> with the pathogenic bacteria and to check the growth of clumped bacteria on the growth medium by evaluating the result with suitable statistical analysis.<\/p>\n

For our convenience Tridax procumbans calyx protein<\/em> has been designated as TPCP.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Plant protein <\/strong><\/p>\n

Calyx of the plant T procumbans L,<\/em> a wild medicinal plant of the family compositeae, was used as the source of plant protein7<\/sup>.<\/p>\n

Reagents and Glassware <\/strong><\/p>\n

Papain, BSA, guar\u2013gum, D \u2013 glucose, D \u2013 galactose, sucrose, raffinose, N \u2013 Acetyl \u2013 D \u2013 glucosamine, D \u2013 glucosamine hydrochloride, a \u2013 pNPG, b – oNPG, Acrylamide, Bis \u2013 acrylamide, b \u2013 mercaptoethanol, coomassie brilliant blue (R 250), were obtained from Sigma chemicals, St Louis, M.O., U.S.A. Distilled water, glass wares like petri dishes, pipettes, glass rods, beakers, test tubes, etc. were sterilized in autoclaved at 121o<\/sup>C at 15 lbs pressure for 15 min. Other chemicals were of analytical grade.<\/p>\n

Bacterial isolates<\/strong><\/p>\n

The bacterial isolates like Enterococci faecalis,<\/em> Escherichia coli,<\/em> Klebsiella pneumonie,<\/em> Morganella\u00a0 species,<\/em> Proteius mirabilis,<\/em> Pseudomonas aeruginosa,<\/em> Providentia species, <\/em>\u00a0Salmonella typhi,<\/em> Salmonella paratyphi- A and<\/em> Salmonella paratyphi-B<\/em> were obtained from Department of Microbiology, Indira Gandhi Government Medical College, Nagpur and were grown on specific growth medium.<\/p>\n

Purification of Plant Protein<\/strong><\/p>\n

Preparation of crude extract<\/strong><\/p>\n

Calyx of 45 days old plants (100 g) grown in the garden of University Department of Biochemistry, Nagpur University, Nagpur, India7<\/sup>, were collected, washed four times under the tap water and two times with distilled water. After soaking between the folds of filter paper, they were homogenized in 1L of 1M NaCl and kept on shaker at 4\u00b0C for 2 hours, to ensure proper extraction of the Plant protein. The extract was passed through two layers of cheese cloth and the filtrate was centrifuged at 12000 rpm (9668g) at 4\u00b0C for 30 min. The supernatant obtained, designated as crude extract, was used for isolation and characterization of TPCP as described elsewhere8<\/sup>.<\/p>\n

Polyacrylamide Gel Electrophoresis<\/strong><\/p>\n

The homogeneity of TPCP was tested on SDS \u2013 PAGE. Molecular weight of the purified plant protein was determined by the method of Weber and Osborn (1969). Acrylamide concentration for the running separator gel was 7.5% and that of the stacking gel was 2.5%, marker proteins such as Carbonic anhydrase 29kD, Trypsin 23kD, Myoglobin 17.2kD, Cytochrome C \u2013 12.3kD, were used as standard proteins. After electrophoresis the gels were stained with 1% coomassie brilliant blue (R \u2013 250) prepared in destaining solution containing 7% acetic acid. The gels were destained in 7% acetic acid9, 10<\/sup>.<\/p>\n

Protein Estimation<\/strong><\/p>\n

Proteins were estimated by the method of Lowry et al.,<\/em> (1951). Bovine serum albumin was used as standard protein (Lowry et. al., <\/em>1951)11<\/sup>.<\/p>\n

Growth Of Bacterial Strains<\/strong><\/p>\n

The pathogenic bacteria were grown on appropriate medium on test tube slants (Table 1).<\/p>\n

Preparation Of The Bacterial Growth Medium<\/strong><\/p>\n

Blood Agar medium<\/strong><\/p>\n

Peptone\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 2%<\/p>\n

Beef extract\u00a0 \u00a01%<\/p>\n

NaCl\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 0.5%<\/p>\n

pH\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 7.4<\/p>\n

Agar\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 3%<\/p>\n

Autoclave upto 560<\/sup><\/p>\n

Add 10% Human or Sheep Blood<\/p>\n

Macconkeys Agar medium<\/strong><\/p>\n

Peptone\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 2%<\/p>\n

Na Taurochlorate\u00a0\u00a0 0.5%<\/p>\n

pH\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 7.4<\/p>\n

Agar\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0\u00a03%<\/p>\n

Red Indicator\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 0.1% aquas solution<\/p>\n

Autoclave upto 560<\/sup><\/p>\n

Add 1% lactose<\/p>\n

Shigella Agar (Ready to use Himedia)<\/strong><\/p>\n

Nutrient Agar medium<\/strong><\/p>\n

Yeast extract\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 5g<\/p>\n

Peptone\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 10g<\/p>\n

pH\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 6.8<\/p>\n

Agar\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 15g<\/p>\n

Distilled water\u00a0\u00a0\u00a0\u00a0\u00a0 1L<\/p>\n

Autoclave upto 560<\/sup><\/p>\n

Bacterial Agglutination<\/strong><\/p>\n

Bacterial agglutination assay using purified TPCP was performed by the method of Zanette, et. al., <\/em>(2000)14<\/sup>. The Bacterial agglutination titre was defined as the reciprocal of the highest dilution that was able to induce visible agglutination12<\/sup>.<\/p>\n

Qualitative determination of clumping of bacteria<\/strong><\/p>\n

This method is a quick screening method for qualitative determination of agglutination. A bacterium was carefully removed from the test tube slants and was suspended in PBS. One drop of 50\u03bcl of heavy bacterial suspension (OD=1) was mixed on a slide with fifty \u03bcl of TPCP (250 \u03bcg per ml), respectively12<\/sup>. Clumping of bacteria with TPCP is considered as positive test. One drop of 50\u03bcl added to 50\u03bcl of normal saline was considered as control.<\/p>\n

Quantitative determination of the clumping of bacteria<\/strong><\/p>\n

The agglutination titre was determined using microtitre plate. The agglutination of bacteria by TPCP was determined by incubating fifty \u03bcl plant proteins (250 \u03bcg per ml) followed by serial dilution. Fifty \u03bcl of bacteria (OD=1) were added to each well separately. After gentle shaking of the reaction mixture (Plant protein + bacteria) was placed on a rotary shaker (50 rpm) for 10 min, the cells were allowed to settle for 45 minutes and the agglutination in the wells were monitored microscopically. Controls were run simultaneously12<\/sup>.<\/p>\n

The Bacterial agglutination titer was defined as the reciprocal of the highest dilution of the T. procumbans<\/em> plant protein extract that yielded visible agglutination activity. One Bacterial agglutination unit (BAU) was defined as the amount of plant protein extract, which causes complete agglutination under the experimental conditions12<\/sup>.<\/p>\n

Table 1:\u00a0Growth medium for pathogenic bacteria<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
No<\/strong><\/td>\nName of microorganism<\/strong><\/td>\nRequired Growth medium<\/strong><\/td>\n<\/tr>\n
1<\/td>\nEnterococci faecalis<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
2<\/td>\nEscherichia coli<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
3<\/td>\nKlebsiella pneumonie<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
4<\/td>\nMorganella\u00a0 species<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
5<\/td>\nProteius mirabilis<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
6<\/td>\nPseudomonas<\/em><\/p>\n

aeruginosa<\/em><\/td>\n

Macconkeys Agar<\/td>\n<\/tr>\n
7<\/td>\nProvidentia<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
8<\/td>\nSalmonella typhi<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
9<\/td>\nSalmonella paratyphi- A<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
10<\/td>\nSalmonella paratyphi-B<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
11<\/td>\nSalmonella entritidis<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
12<\/td>\nSerratia species<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n
13<\/td>\nShigella dysenteria<\/em><\/td>\nShigella Agar<\/td>\n<\/tr>\n
14<\/td>\nStaphylococcus aureus<\/em><\/td>\nBlood Agar<\/td>\n<\/tr>\n
15<\/td>\nVibrio cholera<\/em><\/td>\nMacconkeys Agar<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Growth of clumped bacteria on solid medium<\/strong><\/p>\n

The appropriate agar plates were prepared with specific growth medium as all bacteria were pathogenic and they require specific growth medium. The agar plates were divided into four equal parts. The clumped bacteria by TPCP were determined by incubating fifty \u03bcl lectin (250 \u03bcg per ml) and fifty \u03bcl of bacteria (OD=1) were mixed separately. After gentle shaking, the reaction mixture (lectin + bacteria) was inoculated on agar medium for 24h at 37o<\/sup>C. The bacterial growth was observed on three successive days visually. Controls were run simultaneously13<\/sup>.<\/p>\n

Table 2:\u00a0Characteristics of pathogenic bacteria used for agglutination with TPCPa<\/sup><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Sr. no.<\/strong><\/td>\nName of microorganism<\/strong><\/td>\nAgglutination<\/strong><\/td>\nGram<\/strong><\/p>\n

-ve\/+ve<\/strong><\/td>\n

Cell wall composition<\/strong><\/td>\nResulting infection<\/strong><\/td>\n<\/tr>\n
1<\/td>\nEnterococci faecalis\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n+<\/td>\n–<\/td>\nMannose<\/p>\n

Specific<\/td>\n

Bacillary dysentery<\/td>\n<\/tr>\n
2<\/td>\nEscherichia coli\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n+<\/td>\n–<\/td>\nMannose<\/p>\n

Specific<\/p>\n

require<\/p>\n

Ca+ +<\/td>\n

Urinary tract meningitis<\/p>\n

in children Pneumonia,<\/p>\n

Urinary tract.<\/td>\n<\/tr>\n

3<\/td>\nKlebsiella pneumonie\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n–<\/td>\n–<\/td>\nMannose<\/p>\n

Very large capsule<\/td>\n

Pneumonia<\/td>\n<\/tr>\n
4<\/td>\nMorganella\u00a0 species\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n+<\/td>\n–<\/td>\n<\/td>\nUrinary tract infection<\/p>\n

Wound infection<\/td>\n<\/tr>\n

5<\/td>\nProteius mirabilis\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n+<\/td>\n–<\/td>\n<\/td>\nUrinary tract infection<\/p>\n

Wound infection<\/td>\n<\/tr>\n

6<\/td>\nPseudomonas\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/p>\n

aeruginosa\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n

+<\/td>\n–<\/td>\nGalactose<\/td>\nUrinary tract, wounds,<\/p>\n

Burns, lung infection.<\/td>\n<\/tr>\n

7<\/td>\nProvidentia species\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<\/em><\/td>\n+<\/td>\n–<\/td>\n<\/td>\nDiarrhea<\/p>\n

Urinary tract infection<\/p>\n

Wound infection<\/td>\n<\/tr>\n

8<\/td>\nSalmonella typhi\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n–<\/td>\n–<\/td>\nMannose<\/td>\nTyphoid fever<\/td>\n<\/tr>\n
9<\/td>\nSalmonella paratyphi- A\u00a0 <\/em><\/td>\n–<\/td>\n–<\/td>\nMannose<\/td>\nParatyphoid fever<\/td>\n<\/tr>\n
10<\/td>\nSalmonella paratyphi-B\u00a0\u00a0 <\/em><\/td>\n–<\/td>\n–<\/td>\nMannose<\/td>\nParatyphoid fever<\/td>\n<\/tr>\n
11<\/td>\nSalmonella entritidis\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n–<\/td>\n–<\/td>\nMannose<\/td>\nTyphoid fever<\/td>\n<\/tr>\n
12<\/td>\nSerratia species\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n+<\/td>\n–<\/td>\n<\/td>\nMeningitis\u00a0 Pneumonia,<\/p>\n

 <\/td>\n<\/tr>\n

13<\/td>\nShigella dysenteria\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n+<\/td>\n–<\/td>\nMannose<\/td>\nBacillary dysentery<\/td>\n<\/tr>\n
14<\/td>\nStaphylococcus aureus\u00a0 <\/em><\/td>\n+<\/td>\n+<\/td>\n<\/td>\nBoils carbuncles food poisoning<\/td>\n<\/tr>\n
15<\/td>\nVibrio cholera\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n+<\/td>\n–<\/td>\n<\/td>\nCholera<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

TPCP \u2013 Tridax procumbans calyx protein.<\/em><\/p>\n

 <\/p>\n

Results and Discussion<\/strong><\/p>\n

The TPCP could be purified in good yield on cross-linked guar gum by affinity chromatography8<\/sup>. \u00a0<\/strong>The purified protein exhibited little low molecular weight of 23kD on SDS \u2013 PAGE (Fig. 1 and 2)13<\/sup>. The results show that T. procumbans <\/em>calyx protein was able to agglutinate pathogenic bacteria. The bacterial agglutination titre was least with Vibrio <\/em>cholera and Serratia species<\/em> as compared to other pathogenic bacteria (Table 3).<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 1:\u00a0 Electrophoretic mobilities of standard proteinsandT.procumbans calyx protein(TPCP). Acrylamide concentration. 7.5%, standard proteins\u00a0(a) carbonic anhydrase,\u00a0(b) – Trypsin and T.procumbans calyx protein (TPCP).\u00a0(c) Myoglobin,\u00a0(d) Cytochrome C\u00a0(from top to bottom)<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 2: SDS-PAGE: \u00a0<\/em>(a). T. procumbans calyx protein<\/em> (TPCP), (b). Standard proteins<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Table 3:<\/strong>\u00a0 Agglutination of pathogenic bacteria with TPCPa<\/sup>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
 <\/p>\n

No<\/strong><\/td>\n

 <\/p>\n

Name of organism and Agglutination with TPCP<\/strong><\/td>\n

 <\/p>\n

No.of<\/strong><\/p>\n

Samples<\/strong><\/td>\n

 <\/p>\n

Bacterial<\/strong><\/p>\n

 <\/td>\n<\/tr>\n

Average titre<\/strong><\/td>\nAgglutination Units<\/strong><\/td>\n<\/tr>\n
Crude<\/strong><\/td>\nASFb<\/sup><\/strong><\/td>\nAC<\/strong><\/p>\n

ASFc<\/sup><\/strong><\/td>\n

Crude<\/strong><\/td>\nASF<\/strong><\/td>\nAC<\/strong><\/p>\n

ASF<\/strong><\/td>\n<\/tr>\n

1<\/td>\nEnterococci <\/em><\/p>\n

faecalis\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 +\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n

5<\/td>\n1:2<\/td>\n1:4<\/td>\n1:8<\/td>\n512**<\/td>\n256**<\/td>\n128*<\/td>\n<\/tr>\n
2<\/td>\nEscherichia <\/em><\/p>\n

coli\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 +<\/em><\/td>\n

5<\/td>\n1:2<\/td>\n1:4<\/td>\n1:8<\/td>\n512**<\/td>\n256**<\/td>\n128*<\/td>\n<\/tr>\n
3<\/td>\nKlebsiella pneumonie\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 –\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n5<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n–<\/td>\n<\/tr>\n
4<\/td>\nMorganella species\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 +<\/em><\/p>\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n

5<\/td>\n1:2<\/td>\n1:4<\/td>\n1:8<\/td>\n512**<\/td>\n256**<\/td>\n128*<\/td>\n<\/tr>\n
5<\/td>\nProteius <\/em><\/p>\n

mirabilis\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 +<\/em><\/td>\n

5<\/td>\n1:2<\/td>\n1:4<\/td>\n1:8<\/td>\n512**<\/td>\n256**<\/td>\n128*<\/td>\n<\/tr>\n
6<\/td>\nPseudomonas\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/p>\n

aeruginosa\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 +\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n

5<\/td>\n1:2<\/td>\n1:4<\/td>\n1:8<\/td>\n512**<\/td>\n256**<\/td>\n128*<\/td>\n<\/tr>\n
7<\/td>\nProvidentia <\/em>species\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em>+\u00a0 <\/em><\/td>\n5<\/td>\n1:2<\/td>\n1:4<\/td>\n1:8<\/td>\n512**<\/td>\n256**<\/td>\n128*<\/td>\n<\/tr>\n
8<\/td>\nSalmonella <\/em><\/p>\n

typhi\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 –<\/em><\/td>\n

 <\/p>\n

5<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n<\/tr>\n

9<\/td>\nSalmonella paratyphi- A\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 –<\/em><\/td>\n <\/p>\n

5<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n<\/tr>\n

10<\/td>\nSalmonella paratyphi-B\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 –<\/em><\/td>\n <\/p>\n

5<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n<\/tr>\n

11<\/td>\nSalmonella entritidis\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 –<\/em><\/td>\n <\/p>\n

5<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n

 <\/p>\n

–<\/td>\n<\/tr>\n

12<\/td>\nSerratia\u00a0 species +<\/em><\/td>\n5<\/td>\n1:4<\/td>\n1;8<\/td>\n1:16<\/td>\n256**<\/td>\n128*<\/td>\n64**<\/td>\n<\/tr>\n
13<\/td>\nShigella <\/em><\/p>\n

dysenteria\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 +<\/em><\/td>\n

5<\/td>\n1:2<\/td>\n1:4<\/td>\n1:8<\/td>\n512**<\/td>\n256<\/td>\n128*<\/td>\n<\/tr>\n
14<\/td>\nStaphylococcus aureus\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 +<\/em><\/td>\n5<\/td>\n1:2<\/td>\n1:4<\/td>\n1:8<\/td>\n512**<\/td>\n256<\/td>\n128*<\/td>\n<\/tr>\n
15<\/td>\nVibrio cholera\u00a0\u00a0\u00a0\u00a0\u00a0 +\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/td>\n5<\/td>\n1:4<\/td>\n1:8<\/td>\n1:16<\/td>\n256**<\/td>\n128*<\/td>\n64**<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Control<\/p>\n

Normal saline<\/p>\n

Plant protein only.<\/p>\n

*p <= 0.001, **p <=0.02, ***p<=0.05.<\/p>\n

TPCP \u2013 Tridax procumbans<\/em> calyx protein.<\/p>\n

ASF \u2013 Ammonium sulphate fraction.<\/p>\n

AC-ASF \u2013 Affinity column Ammonium sulphate fraction.<\/p>\n

 <\/p>\n

Experiments were run in triplicates<\/strong><\/p>\n

The characteristics of bacterial strains that specifically were agglutinated by TPCP are shown in Table 2 and 3; Fig. 3. TPCP proved to be effective agglutinating agent for Staphylococcus aureus<\/em>,<\/em> Enterococci<\/em> faecalis,<\/em> Escherichia coli, <\/em>Pseudomonas<\/em> species, Shigella species, Proteius mirabilis, Morganella species<\/em> and Providentia species.<\/em> Whereas, Salmonella species<\/em> and Klebsiella species<\/em> were unable to agglutinate with TPCP. The bacterial strains gave positive agglutination after 4th<\/sup> serial dilution. The strains of Serratia<\/em> and Vibrio<\/em> cholera<\/em> required lowest titre for agglutination as compared to the other bacteria (Table 3; Fig. 3).<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 3:\u00a0Agglutination of pathogenic bacteria with TPCP (microtiter plate showing carpet and button).\u00a0a) Tridax procumbans calyx protein<\/em> (TPCP).\u00a0b) Positive control (Plant protein + O positive group erythrocytes) and Negative control (Plant proteins only).<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

The clumped bacteria by the extract of Tridax procumbans L. <\/em>did not show growth on solid medium (Fig. 4).<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 4:\u00a0Determination of the growth on solid medium: The clumped bacteria by the extract of TPCP did not show growth on the agar medium.\u00a0Positive control (Plant protein + Bacteria) and Negative control (Plant proteins only).\u00a0Agar Slants;\u00a0<\/span>Agar plates.<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

The agglutination of bacteria with TPCP indicates that these bacteria carry the receptor sites. This could be recognized by TPCP and bind with the bacteria thereby forming clumps.<\/p>\n

The binding properties and specificities of TPCP may have practical benefit; and it can be used to classify the bacteria, which can serve as an additional test for identification of several pathogenic bacteria. For instance, these studies could be used as a screening test for Salmonella species and Klebsiella<\/em> that do not have the property to agglutinate with TPCP. In addition, the studies may serve as the basis of a novel therapeutic approach14<\/sup>. Plant proteins are able to selectively clump Pyogenic cocci<\/em>, similarly, groups A, B, C, F and G streptococci can be readily distinguished. Salmonella genus<\/em> was unable to react with Con A. The Shigellae<\/em> are simpler from classification viewpoint because they do not possess flagellar H-antigens and consists of four species. Of the four main Shigella<\/em> species tested, only Shigella flexneri<\/em> was readily clumped by Con A. Shigella boydii<\/em> and Shigella dysenteriae<\/em> reacted only slowly with the lectin Con A suggesting that it is possible that lectins could classify Enterobacteriaceae<\/em>, Salmonella<\/em> genus and Shigella species <\/em>8<\/sup>.<\/p>\n

The results presented here indicate that the probable specific receptor for TPCP that agglutinated pathogenic bacteria and obstruct their growth by blocking the surface receptors. Thus this extract can be used for the treatment of bacterial dysentery or for preparation of creams for topical application on wounds to prevent bacterial invasion of tissues.<\/p>\n

Acknowledgements<\/strong><\/p>\n

The authors thank Dr. Jalgaonkar, Head, Department of Microbiology, IGGMC Nagpur, for providing bacterial strains and grateful to the Head, Department of Biochemistry, R. T. M. Nagpur University, Nagpur for encouragement.<\/p>\n

Precautions<\/strong><\/p>\n

Safe working and the prevention of infection was strictly followed according to Health Services Advisory Committee (2003).<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Nilsson C., Lectins: Analytical Tools from Nature.; Lectins, Pages 1-13, (2007).<\/li>\n
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  3. Reetz, M., Zonta, A. and Simpelkamp, J., Efficient immobilization of lipases by entrapment in hydrophobic sol-gel materials. Bioeng.,<\/em> 49: 527-534, (1996).<\/li>\n
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  7. Kohler, W. and Prokop, O., Agglutinins versuchean Streptokokken mit dem Phytoagglutinin and Dolichos biflorus<\/em>. Immunitaetsforsch Allerg . Klin. Immunol.,<\/em> 133: 171-175, (1967).<\/li>\n
  8. Ramteke A., P. and Patil M., B., Purification and Characterization of Tridax procumbans<\/em> calyx lectins. Biosci. \u00a0 Res. Asia. 3(1), 103 \u2013 110, (2005).<\/li>\n
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  11. Davis, B, J., Annals of New York Academy of Sciences.<\/em>, 121: 404 \u2013 427, (1964).<\/li>\n
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  13. Zanette, D., Sarubbi, E.,\u00a0G., Soffientini, A. and Restelli, E., Process for the Purification of Glycoproteins like Erythropoietin. European Patent Office<\/em>, EP 0 820 468 B1, (2000).<\/li>\n
  14. Goto, M. Hikota, T., Nakajima, M., Kakikawa, Y. and Tsuyumi, S., Occurance and properties of copper resistance in plant pathogenic bacteria. Annu. Soc. Japan., 60: 147-153, (1994).<\/li>\n
  15. Mulvey, G., Kitov, P., , Marcato, P., Bundle, D., R. and Armstrong, G., D. Glycan mimicry as a basis for novel anti-infective drugs. Biochimie., <\/em>83: 841\u2013847, (2001).<\/li>\n
  16. Health Services Advisory Committee; Safety in Health Service Laboratories, Safe working and the prevention of infection in clinical laboratories and similar facilities. 2nd ed. Suffolk: H. S. E., Books; (2003).<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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