Mythreyi N et al. / Journal of Pharmacy Research 2011,4(11),
Research Article ISSN: 0974-6943
Available online through www.jpronline.info
Salt Aggregation Test and Hemagglutination Assay for Understanding Bacterial Adherence Mythreyi N, Rathina Kumar S, Sriram K, Pavithra M, Asit R Ghosh*. Centre for Infectious Diseases and Control, VIT University, Vellore, India
Received on: 19-05-2011; Revised on: 08-06-2011; Accepted on:01-07-2011 ABSTRACT For the initiation of any microbial infection, the first and foremost step is the adherence of microbes to the host. Hence to study the pathogenesis of the microbe, we must have knowledge on the molecular mechanisms of adherence of microbes to the host cell. Hemagglutination (HA) assay and Salt Aggregation Test (SAT) can be used as very simple and rapid methods for studying the adherence of bacterial cells. In this study, certain prototype microbes like Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25619), Staphylococcus aureus (ATCC 25923), Lactobacillus gasseri (ATCC 19992), Pediococcus spp.GS4, Klebsiella spp., Listeria spp. and 20 isolated E.coli strains were studied using HA assay and SAT. Of the 7 organisms only E.coli showed HA and only one of the 20 strains of E.coli isolated from food sources in a separate study, showed HA. All organisms showed SAT positive at all concentrations except L.gasseri (ATCC 19992). Of the 20 E.coli strains, only 8 showed SAT positive at all concentrations. The different sugars did not have any effect on the hemagglutination of E.coli (ATCC 25922) and it showed HA in all human blood groups but not in bovine erythrocytes. Combined SAT and HA are found to be simple, rapid and informative to draw a general conclusion on adherence of any culturable bacteria including pathogens at peripheral laboratories. Key words: Surface hydrophobicity, SAT, HA
INTRODUCTION The prerequisite for bacterial colonization or infection is the adherence of the organism to the host cell [5]. Adhesion of bacteria is not only governed by long range forces such as steric and electrostatic interactions, but also by short range forces such as hydrophobic interactions, vanderwaals, acid-base, hydrogen bonding and biospecific interactions. The bio-specific adherence occurs between the structural components of the bacteria like fimbriae, adhesins or hemagglutinin and glycoproteins of the cell [3]. Many organisms have the ability to adhere to the human epithelial cells leading to various diseases. Since the surface structure of RBC resembles that of epithelial cells, it can be used as a prototype cell to study adherence using hemagglutination [2]. Hemagglutination (HA) is the aggregation of red blood cells in the presence of bacteria. The bacteria and RBCs crosslink together and form visible clumps. This occurs because of the substances called hemagglutinin present on the bacterial cell surface. These hemagglutinins are capable of binding to certain carbohydrate residues in the erythrocyte cell surface. The haemagglutinins present in the bacteria may be lectins or N-acetyl neuraminidase. Based on the presence or absence of HA, the presence or absence of the Hemagglutinin can be found out. Here we concentrate on the carbohydrate binding proteinslectins for bacterial adhesion [12]. Based on their binding affinity, lectins are of so many types. For example, in urinary tract infection, lectins bind to the cell surface at the carbohydrate residues. Bacterial adhesion to host cells is also governed by non specific interactions such as cell surface hydrophobicity [14]. Hydrophobicity plays a vital role in the adherence of microbes to a wide variety of surfaces such as skin, soft tissue, wounds and endothelial cells and facilitates biofilm formation due to bacterial adhesion. The cell surface hydrophobicity (CSH) is expressed by many compounds such as the fimbriae of Gram-negative bacteria such as E.coli, the carbohydrate capsule of S.aureus and the lipotechoic acid of Gram-positive bacteria [10]. This surface hydrophobicity can be measured as the affinity of binding to amphiphillic gels or the tendency to autoaggregate in the presence of salt solutions such as ammonium sulphate [ 9 ]. The autoaggregation phenomenon is similar to that of the salting out process. The latter is used in the Salt Aggregation Test (SAT). In this study, HA and SAT were carried out to demonstrate the adherence property of some prototype bacteria which are regarded as pathogens including environmental (Food) isolates to validate the hypothesis that adherence is pre-requisite to pathogenicity or colonization (biofilm formation). MATERIALS AND METHODS: Bacterial strains used: E. coli(ATCC 25922), P s e u d o m o n a s a e r u g i n o s a ( A T C C 2 5 6 1 9 ) , Staphylococcus aureus(ATCC 25923), Lactobacillus gasseri (ATCC 19992),
*Corresponding author. Asit R Ghosh*. Professor & Asst. Director Centre for Infectious Diseases and Control, VIT University, Vellore – 632 014, India Tel.: 0416-2202618.09790238701 E-mail:
[email protected], Website: vit.ac.in
Pediococcus spp.GS4, Klebsiella spp., Listeria spp. and 20 isolated E.coli strains. Preparation of Blood sample: Human blood group types- O+, O-,A +, B+ and bovine blood were obtained.4.5ml of blood was collected in a tube containing 0.5ml of 3.8% citric acid. It was centrifuged at 5000rpm for 10min in a cooling centrifuge. The pellet was dissolved in 10ml of PBS and the centrifugation was repeated. The pellet is then suspended in 5ml of PBS and stored in refrigerator [1]. Preparation of Bacterial suspension: 1ml of each culture was centrifuged at 10,000 rpm for 10min. The pellet was suspended in 1ml of PBS and the centrifugation step was repeated once again. The pellet is then suspended in 100µl of PBS. Salt aggregation test (SAT): The SAT for surface hydrophobicity was performed using different molar concentrations of ammonium sulphate [8]. Ten microlitres of bacterial suspension was mixed with equal volume of ammonium sulphate of varying molarity (0.01M, 0.05M, 0.1M, 0.15M, and 0.2M) on a glass slide and observed for aggregation after 1 min at 20 oC. Hemagglutination (HA): HA was performed using each blood group type against each bacterial suspension. 20µl of bacterial suspension was added to 20µl of erythrocyte suspension (1:3 dilutions) on a microscopic glass slide and rocked well. The reactions were seen visually or through a colony counter lens. The mannose sensitivity and effect of other sugars like glucose, fructose, lactose, sucrose and starch on HA was tested by adding 20µl of 0.5% of sugar to 20µl of erythrocytes before adding bacterial suspension [1]. RESULTS: SAT: Result shown in Table 1 demonstrate the aggregation at the lowest concentration of 0.01M (NH4) 2SO4 by all the strains except L.gasseri (ATCC 19992). With the increase of concentration to the tune of 1.0 M, all the strains were aggregative. With the 20 isolated E.coli strains, E.coli 1, 3, 8, 9, 10, 11, 12, 16, 17, 18 and19 shows aggregation at lower concentrations of 0.01 and 0.05M. E.coli 7, 14 and 15 shows no aggregation at all. E.coli 2, 4, 5, 6, 7, 13 and 20 shows aggregation at higher concentrations. HA: Table 2 shows the result of HA. Only E.coli(ATCC 25922) and E.coli 2 shows HA with all blood samples taken except that of bovine. All others do not show HA with the blood samples taken for examination. Also there is no effect of sugars on HA of E.coli(ATCC 25922) with blood samples. Hence the HA is resistant to sugars.
Journal of Pharmacy Research Vol.4.Issue 11.November 2011
Mythreyi N et al. / Journal of Pharmacy Research 2011,4(11), Table 1: Salt aggregation Test(SAT) Organisms E.coli (ATCC 25922) P. aeruginosa (ATCC 25919) S.aureus (ATCC 25923) Pediococcus spp.GS4 L.gasseri (ATCC 19992) Listeria spp. Klebsiella spp.
Ammonium Sulphate Concentrations(M) 0.01 0.05 0.10
0.15
0.20
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ _
+ _
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
Thus pathogenicity of E.coli can be identified indirectly using hemagglutination assay. The HA assay performed with isolated E.coli strains indicated that 1 out of the 20 strain could be pathogenic. Other microbes such as Listeria spp., L.gasseri(ATCC 19992), S.aureus(ATCC 25923), P.aeroginosa (ATCC 25619), Pediococcus spp.GS4, Klebsiella spp. did not show hemagglutination. It does not mean that they do not infect humans or do not adhere to the site of colonization. But they possibly use other different mechanisms for their adherence [11]. Also adherence mechanism varies for different microbes to different hosts.
Table 2: Hemagglutination(HA) Organisms
Blood Samples O+
O-
A+
B+
Bovine
E.coli (ATCC25922) P.aeruginosa (ATCC 25919) S.aureus (ATCC 25923) Pediococcus spp.GS4 L.gasseri (ATCC 19992)) Listeria spp. Klebsiella spp.
+
+
+
+
_
_
_
_
_
_
_
_
_
_
_
_ _
_ _
_ _
_ _
_ _
_ _
_ _
_ _
_ _
_ _
DISCUSSION: Most of the organisms taken for study showed surface hydrophobicity as revealed by SAT between the concentrations of 0.01-0.05M of ammonium sulphate. Adherence of bacteria to the host cell is the primary step for any infection to occur. The cell surface hydrophobicity plays a significant role in early stages of adherence of the organisms. Hence there is a possibility of these organisms to bind to the host cell through surface hydrophobicity. The hydrophobicity may be contributed by appendages like fimbriae or non- fimbrial surface proteins. In organisms showing aggregation only at higher concentration, non-fimbrial proteins might alone be responsible for aggregation [7]. For example, in gram positive bacteria, teichoic acid in the cell membrane had a conserved sequence of hydrophobic amino acids. Those which do not show SAT positive lack surface hydrophobicity. Hence SAT can be used to confirm the hydrophobic interaction between host and pathogens. Hemagglutination occurs due to the interaction between the carbohydrate binding proteins present on fimbriae and the proteins present on the surface of erythrocytes. There are 6 different types of fimbriae. Not all fimbriae types show hemagglutination. Type 1 and type 3 fimbriae, most commonly seen in microbes show mannose sensitive and mannose resistant hemagglutination respectively. Type 4 fimbriae have hemagglutinating activity only with fresh RBC [13]. So absence of hemagglutination does not indicate the absence of fimbriae [4].Further tests are to be carried out to find out if other types of non-hemagglutinating fimbriae are present. E.coli strains are known to contain either type 1 or type 3 fimbriae and not the other types. In the experiment perfomed, E.coli strain (ATCC 25922) showed agglutination with human RBC but not with bovine RBC. When tested for the effect of various sugars on hemagglutination of E.coli, hemagglutination was found even in the presence of sugars like glucose, lactose, mannose, fructose, galactose, starch and sucrose. This indicates the presence of type 3 fimbriae and it could be an instance of Mannose Resistant Hemagglutination (MRHA). MRHA is directly related to urinary tract infection [15]. Also it indicates that this E.coli (ATCC 25922) can bind to epithelial cells of human urinary tract and cause infection but cannot infect the urinary tract of bovine as it does not show agglutination.
CONCLUSION: For studying host-microbe interaction epithelial cells are used previously. But HA will be proved to be effective than using epithelial cells directly because processing of epithelial cells include isolation from the gut, purification, removal of contaminants etc (16). Here RBC is a surrogate cell to epithelial cells. Since the morphology of epithelial cells and RBCs are similar, HA can be a simple and instantaneous method for the primary detection of the pathogenicity of organisms such as E.coli. Combined SAT and HA are found to be simple, rapid and informative to draw a general conclusion on adherence of any culturable bacteria including pathogens at peripheral laboratories. REFERENCES : 1.
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Source of support:VIT, University, Vellore. ;Conflict of interest: None Declared
Journal of Pharmacy Research Vol.4.Issue 11.November 2011
