多重实时荧光定量PCR检测肠出血性大肠杆菌O157:H7

目的 利用多重荧光定量PCR技术,建立一种快速、准确、特异检测肠出血性大肠杆菌O157:H7的定量方法.方法 选取肠出血性大肠杆菌O157:H7编码脂多糖基因(rfbE)和编码鞭毛抗原基因(fliC)作为检测的靶基因,设计引物和TaqMan-MGB探针,探针的5'端分别用FAM和HEX进行荧光标记,3'端标记MGB.优化PCR扩增体系,对多重实时荧光定量PCR方法的特异性、灵敏度、重复性评价,同时进行一定数量临床样本鉴定,与常规方法进行比较.结果 本研究所建立的多重实时荧光定量PCR方法可准确、特异地检测和鉴定肠出血性大肠杆菌O157:H7,能够有效甄别肠出血性大肠杆菌O157:H7与非H7菌株,其他菌株均无阳性结果;该方法的灵敏度可达到10 CFU/ml;定量检测的批间和批内变异系数均小于5%;对66例临床样本进行评价,结果显示15例肠出血性大肠杆菌O157:H7阳性,2例为肠出血性大肠杆菌O157:非H7阳性,其中16例与常规培养法结果符合,符合率达到98.49%.结论 本研究建立的检测肠出血性大肠杆菌O157:H7多重实时荧光定量PCR方法快速,结果准确、可靠,操作简便,为肠出血性大肠杆菌O157:H7的临床诊断、现场流行病学调查和食品安全监测提供了新的鉴定方法.     

肠出血性大肠杆菌O157:H7特异性脂多糖抗原的制备

肠出血性大肠杆菌（Enterohemorrhagic Escherichia coil，EHEC）O157：H7是产Veto毒素的食源性肠道致病菌，可引起人类患血性腹泻、肾衰竭、尿毒综合征等症。本文应用热酚水法提取纯化了EHEC0157：H7脂多糖抗原及其O-特异性多糖（O-specific polysaocharide，O-SP）侧链。     

肠出血性大肠杆菌O157:H7溶血素保护性片段(HlyAN436)克隆、表达、初步纯化与生物学活性研究

目的获取重组表达肠出血性大肠杆菌O157的溶血素（Ehx）保护性片段，并研究其基本的生物学及免疫学特性。方法PCR法从EHECO157-SMR2菌株克隆O157：H7的HlyA亚单位的N端片段（436个氨基酸），T-A克隆后构建表达载体pET-28a（＋）-HlyAN436，测序鉴定后转化到E.coliBL21（DE3），IPTG诱导表达，初步纯化后免疫动物。结果Hly-AN436成功地在大肠杆菌表达系统表达，动物实验证实其具有良好的免疫原性和反应性以及一定的免疫保护性。结论Hly-AN436蛋白有可能用于肠出血性大肠杆菌O157基因工程疫苗的研究。     

抗出血性大肠杆菌O157:H7单克隆抗体的制备及初步应用

1983年美国Riley等~([1])首次报道肠出血性大肠杆菌O157:H7,我国于1987年首次在出血性肠炎患者体内分离到大肠杆菌O157:H7.     

多重PCR-DHPLC技术检测肠出血性大肠杆菌O157:H7基因型的方法学研究

目的 开发肠出血性大肠杆菌O157:H7的多重PCR-变性高效液相色谱(DHPLC)检测方法 .方法 以编码大肠杆菌0157抗原的rfbE 基因、编码毒力因子的类志贺毒素(SLT)基因为目的 基因,选择2对引物,建立并优化了大肠杆菌O157:H7的多重PCR-DHPLC检测体系.扩增产物分别为224 bp和499 bp.结果 采用37株细菌验汪了该多重PCR具有良好的特异件.PCR榆测的灵敏度可达到4 CFU/ml.结论 实验证明,研究建立的多重PCR-DHPLC方法 可特异、灵敏地实现对大肠杆菌O157:H7的检测.     

肠出血性大肠杆菌O157∶H7志贺毒素I型A亚基的表达与鉴定

目的表达肠出血性大肠杆菌(EHEC)O157∶H7志贺毒素1型A亚基(Stx1A)重组蛋白并鉴定。方法从EHEC O157∶H7基因组中扩增编码Stx1A的基因,经测序无误后,克隆入表达载体pET-22b(+),转化大肠杆菌BL21(DE3),经诱导、纯化获得目的蛋白Stx1A,并对纯化的目的蛋白进行质谱鉴定。重组的Stx1A蛋白免疫BALB/c小鼠,观察小鼠抗血清与EHEC O157∶H7毒株特异性反应。结果 PCR扩增的Stx1A基因为945 bp,成功构建重组质粒pET22b(+)-Stx1a,重组蛋白在原核细胞中获得高效表达,通过AKTATM-His亲和层析柱获得纯化。质谱分析表明目的蛋白为Stx1A。Western blot显示鼠抗Stx1A血清可与EHEC O157∶H7毒株产生的天然毒素蛋白结合。结论成功克隆了EHEC O157∶H7 Stx1A基因,并获得重组表达,为后续研究奠定基础。     

大肠杆菌O157：H7多糖—重组铜绿假单胞菌外毒素A结合疫苗的研制

目的用大肠杆菌O157∶H7(Escherichia coli O157∶H7, E. coli O157)的一个重要保护性抗原即菌体脂多糖(LPS)制备有效的候选疫苗. 方法选用Westphal热酚法提纯E. coli O157∶H7的LPS,经酸水解法进一步脱毒处理后得到无毒但却具弱免疫原性的O-特异性多糖(O-SP),然后采用己二酸二肼(ADH)将其共价结合到琥珀酰化(或没琥珀酰化)重组铜绿假单胞菌外毒素(rEPAsucc或rEPA)上. 结果制备得到的E. coli O157∶H7的O-SP-rEPAsucc或O-SP-rEPA结合疫苗是安全有效的,其免疫原性要比单一O-SP的免疫原性强,免疫小鼠后产生的血清抗LPS IgG抗体滴度比单一O-SP免疫后产生的抗体水平均有显著性升高(P<0.01),且再次注射后存在加强应答. 结论 E. coli O157∶H7的O-SP-rEPAsucc结合疫苗可望作为一种预防肠出血性E. coli引起的感染性腹泻的有效候选用疫苗.     

产志贺毒素大肠杆菌O157∶H7的亚碲酸钾抗性基因簇分布和抗性水平

目的 了解产志贺毒素大肠杆菌O157：H7的亚碲酸盐抗性基因簇的分布和抗性水平的关系。方法运用PCR和核酸杂交方法进行基因检测，使用平皿法检测亚碲酸钾抗性水平。结果在所检测的志贺菌、沙门菌、霍乱弧菌、耶尔森菌、泌尿道致病性大肠杆菌、肠产毒性大肠杆菌、肠致病性大肠杆菌、肠侵袭性大肠杆菌、肠黏附性大肠杆菌、肠出血性大肠杆菌中，只有大肠杆菌O157：H7具有ter(tellurite resistance，ter)基因簇。所检测的34株产志贺毒素的大肠杆菌O157：H7有33株具有ter基因簇，所检测的18株不产生志贺毒素的大肠杆菌O157，4株具有ter基因簇，但是没有该毒力岛的其它基因。ter基因阳性的33株菌中，亚碲酸钾的抗性水平一般较高，4株在750μg/ml以上，19株为500～750μg/ml，10株为250～500μg/ml；而4株ter基因簇阴性的菌株，也表现了高水平的耐药性，其抗性水平均为500～750μg/ml。其它ter基因簇阴性的菌株，20株耐药性为5～10μg/ml，1株10～20μg/ml，6株20～50μg/ml，2株50～100μg/ml，1株100～250μg/ml。结论ter基因簇在产志贺毒素的大肠杆菌O157：H7中高频率存在，而在其它病原菌中没有检测到。高水平的亚碲酸钾抗性可以作为选择性分离产志贺毒素大肠杆菌O157：H7的指标之一。     

肠出血型大肠埃希菌O157：H7 EspF蛋白多克隆抗体的制备和初步纯化

目的制备肠出血型大肠埃希菌（EHEC）O157：H7EspF蛋白多克隆抗体并初步纯化。方法生物信息学方法分析EHECO157：H7EspF蛋白的柔韧性、亲水性、表面可能性及抗原表位,选取1条由15个氨基酸残基组成的多肽为半抗原,在其C端偶联钥孔血蓝蛋白（KLH）,免疫新西兰大白兔制备抗血清。ELISA测定抗血清效价,免疫印迹法鉴定其特异性,辛酸-硫酸铵法初步纯化抗血清,SDS-PAGE检测抗体纯度。结果选择EspF蛋白抗原指数最高的肽段72-TPSRPAPPPPTSGQA-86（0.506）为半抗原,合成抗原多肽经高效液相色谱鉴定,纯度为95.78%,经质谱分析其分子量为1563.76Mr,与目的多肽分子量一致;加强免疫3次后,EHECO157：H7EspF蛋白的抗血清效价达1：2048000;该血清对EHECO157：H7野生株和espF突变株（△espF）的EspF蛋白均有特异性,并经辛酸-硫酸铵法获得一定纯度的抗体。结论成功地制备了效价高、特异性强的EHECO157：H7EspF蛋白多克隆抗体。     

抗肠出血性大肠杆菌O157:H7 EspA单克隆抗体的制备及其特性鉴定

目的:制备抗肠出血性大肠杆菌O157:H7EspA单克隆抗体(mAb)。方法:以重组EspA蛋白为免疫原免疫BALB/c小鼠,运用杂交瘤技术并且联合间接ELISA筛选只针对EspA的杂交瘤细胞株。以Ig类与亚类鉴定试剂盒鉴定mAb的Ig亚类,ELISA、Western blot及免疫荧光技术鉴定抗体的免疫学特性。结果:获得3株稳定分泌抗EspA杂交瘤细胞的mAb,Ig亚型分别为IgG1κ型、IgG2aκ型、IgG2bκ型。Western blot和免疫荧光分析表明,3株杂交瘤细胞制备的mAb腹水都能特异地结合重组EspA蛋白和识别O157:H7的EspA成分。结论:成功地制备了抗肠出血性大肠杆菌O157:H7EspA的mAb,并证明了该mAb的生物学活性,为深入研究肠出血性大肠杆菌O157:H7的致病机制提供新的手段。     

Development of a monoclonal antibody-based ELISA to detect Escherichia coli O157:H7

A pair of monoclonal antibodies (mAb) from 10 murine hybridomas secreting Escherichia coli O157:H7 (E. coli O157:H7)-specific mAbs were selected for the development of the sandwich ELISA to detect E. coli O157:H7. On the basis of pairwise interaction analysis, mAb-1 was selected as a capture antibody while mAb-6 was used as a detection antibody. The buffer system which provided the greatest difference between the specific E. coli O157:H7-positive antigen and the negative control was chosen. This sandwich ELISA showed good linearity when the concentration of E. coli O157:H7 was in the range of 10⁵–10⁸ cfu/mL, and the sensitivity was 1×10⁴ cfu/mL. With 8-h enrichment of bacteria, this ELISA was found to detect 0.4 cfu/g E. coli O157:H7 in artificially contaminated green tea samples.     

Variation in the persistence of Escherichia coli O157:H7 in experimentally inoculated 6-week-old conventional lambs

Six-week-old lambs were inoculated orally with 10(9) cfu of an antibiotic-resistance marked four-strain mixture of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 to investigate faecal excretion and intestinal colonisation. In the first experiment, three E. coli O157:H7 isolates were not detected in the faeces of any lambs beyond day 8 post inoculation (pi), or from any of the tissues derived from inoculated animals. One strain, 140065 Nal(r), was isolated from the caecum and colon of one lamb on day 9 pi, from the rectum of another on day 22 pi and persisted in the faeces for up to 28 days pi. All animals remained clinically normal throughout the study period and histological evidence of adhesion of E. coli O157:H7 to the intestinal mucosa was not found. In a separate experiment, four 6-week-old lambs were inoculated orally with 10(9) cfu of E. coli O157:H7 strain 140065 Nal(r) alone. Faecal samples were positive for this strain until the end of the experiment (day 19 pi). This strain was also recovered from the gastrointestinal tract of lambs on days 6, 18 and 19 pi, but was not isolated at day 17 pi. When sampled separately, rectum and terminal colon contents contained higher numbers of the inoculated strain than the intestinal tissue at these sites. Animals inoculated with O157:H7 strain 140065 Nal(r) alone produced soft faeces from day 5 pi onwards. Although attaching and effacing lesions were observed in the caecum, proximal colon and rectum in one animal on day 18 pi, the adherent bacteria did not stain with antiserum raised against the O157 antigen.     

Detection of Escherichia coli O157:H7 using chicken immunoglobulin Y

A sandwich ELISA technique was examined to detect Escherichia coli O157:H7 using chicken anti-E. coli O157:H7 IgY as the capture-antibody and an anti-E. coli O157 mouse mAb conjugated with biotin as the detection antibody. The anti-E. coli O157:H7 IgY was harvested from eggs laid by hens (23 weeks of age, Single Comb White Leghorn) immunized with formalin-killed E. coli O157:H7. The IgY was purified by water dilution methods and gel chromatography on Sephacryl S-300 followed by ammonium sulfate precipitation. The sensitivity (CFU/ml) of sandwich ELISA for the E. coli O157:H7 was repeatedly examined with 10 replicates of each sample and a standard curve was plotted. The sandwich ELISA can detect as low as 40CFU/ml of E. coli O157:H7. The data suggest that chicken IgY-based sandwich ELISA provides a reliable, inexpensive and sensitive assay for the detection of the food-borne pathogen E. coli O157:H7.     

Development of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies to O157 Antigen of Escherichia coli

The O157 antigen of Escherichia coli shares structural elements with lipopolysaccharide (LPS) antigens of other bacterial species, notably Brucella abortus and Yersinia enterocolitica O9, a fact that confounds the interpretation of assays for anti-O157 antibodies. To address this problem, a blocking enzyme-linked immunosorbent assay (bELISA) was designed with E. coli O157:H7 LPS as the antigen and a monoclonal antibody specific for E. coli O157, designated 13B3, as the competing antibody. The bELISA had equivalent sensitivity to, and significantly higher specificity than, the indirect ELISA (iELISA), detecting anti-O157 antibodies in sera from cattle experimentally inoculated with O157:H7. Only 13% of sera from naive heifers vaccinated for or experimentally infected with B. abortus had increased anti-O157 bELISA titers, while 61% of anti-O157 iELISA titers were increased. The bELISA is a sensitive and specific method for the detection of serum antibodies resulting from exposure to E. coli O157.     

Serodiagnosis by passive hemagglutination test and verotoxin enzyme-linked immunosorbent assay of toxin-producing Escherichia coli infections in patients with hemolytic-uremic syndrome.

Eight cases of hemolytic-uremic syndrome in which no pathogens were isolated were diagnosed serologically by a passive hemagglutination assay and a verotoxin (VT; Shiga-like toxin) enzyme-linked immunosorbent assay (ELISA). The passive hemagglutination assay employed formalinized sheep erythrocytes sensitized with soluble native antigen or heat-treated antigen (lipopolysaccharide [LPS]) from Escherichia coli O26, O111, O128, and O157 or flagellar antigen of nine different H serogroups of E. coli: H2, H7, H8, H10, H11, H12, H18, H19, and H25. All patients had antibodies against the native antigen and/or the LPS of E. coli O157, but positive agglutination with H7 was observed only in one patient. In the VT-ELISA with plates coated with purified VT 1 or VT 2, antibody against VT 2 was observed in the sera of five of six patients examined, but none of the patients possessed VT 1 antibody. These results indicate that the causative pathogen in these eight hemolytic-uremic syndrome cases is likely to be VT-producing E. coli O157. The passive hemagglutination assay described here is a very sensitive, simple, and rapid method. This assay is highly recommended for the serological diagnosis of VT-producing E. coli infections, particularly in patients infected by serogroup O157 strains. Furthermore, the VT-ELISA is useful in studying the role of VT in hemolytic-uremic syndrome.     

Strains of Escherichia coli O157:H8 from human diarrhoea belong to attaching and effacing class of E coli.

AIMS: To determine whether 17 Escherichia coli O157:H8 strains isolated from patients with diarrhoea in the United Kingdom were putative pathogens. METHODS: The strains had been isolated by the use of O157 antiserum, available for the detection of Vero cytotoxin (VT) producing strains of E coli O157 that are usually of flagellar (H) type 7, but may also be non-motile. The strains were examined for VT production, for their ability to adhere to HEp-2 cells, and for hybridisation with several DNA probes that recognise pathogenic properties of E coli. Their ability to ferment sorbitol and to produce beta-glucuronidase was also investigated, as these tests are used to discriminate VT positive O157 strains. RESULTS: The O157:H8 strains did not produce VT. All gave localised attachment to HEp-2 cells, associated with a positive fluorescence-actin staining test, and all hybridised with the E coli attaching and effacing (eae) probe. In addition to the difference in VT production, O157:H8 strains could be distinguished from VT positive O157 strains by their beta-glucuronidase activity, their failure to produce enterohaemolysin, and their lack of hybridisation with the CVD419 probe derived from a plasmid in an O157:H7 strain. CONCLUSIONS: The 0157:H8 strains had in vitro properties characteristic of the class of E coli that causes attaching and effacing lesions in epithelial intestinal cells. They may therefore be considered a putative cause of diarrhoea but their prevalence remains to be established. Several O157:H8 strains failed to ferment sorbitol in agar plates and therefore could be misidentified as VT positive O157 strains. Confirmatory tests for VT production are needed when O157 strains are isolated from faeces.     

H7 antiserum-sorbitol fermentation medium: a single tube screening medium for detecting Escherichia coli O157:H7 associated with hemorrhagic colitis.

Escherichia coli serotype O157:H7 has been isolated from outbreaks and sporadic cases of hemorrhagic colitis. There is convincing evidence that it can cause this diarrheal disease. Because of the interest in hemorrhagic colitis, it has become desirable to detect this particular strain in human feces, which usually contains many other strains of E. coli. Two characteristics of the incriminated E. coli O157:H7 strain have made its isolation and identification easier. It does not ferment D-sorbitol rapidly, in contrast to about 95% of other E. coli strains. In addition, the strain has H antigen 7, but only about 10% of other E. coli strains have this particular antigen. To screen for E. coli O157:H7 we devised H7 antiserum-sorbitol fermentation medium (18 g of enteric fermentation base, 10 g of D-sorbitol, 4 g of agar, 10 ml of Andrade indicator, 989 ml of water; all ingredients were mixed, autoclaved, and cooled; 1 ml of E. coli H7 antiserum was then added). Colonies to be screened were inoculated into this medium. Strains of E. coli O157:H7 gave a characteristic pattern; they did not ferment sorbitol and were immobilized in the semisolid medium because of the reaction of their flagella with the flagella antiserum. Almost all other strains of E. coli gave a different pattern; they fermented sorbitol or were not immobilized by the H7 serum or both. Strains which were presumptive positives (sorbitol negative, H7 positive) were then tested in E. coli O157 serum by slide or tube agglutination. The number of strains which were presumptive positive by H7-sorbitol medium but then were not found to be O157 was less than 1%. A second approach has been helpful in deciding which colonies to screen in H7-sorbitol medium. MacConkey-sorbitol agar (22.2 g MacConkey agar base [which contains no sugar], 10 g of D-sorbitol, 1,000 ml of water) was designed as a plating medium. Stools were plated on MacConkey agar to estimate the number of E. coli colonies and also plated on MacConkey-sorbitol agar to estimate the number of sorbitol-negative colonies of E. coli. These two approaches have proved useful for isolating and identifying E. coli O157:H7 form human feces and from feces of animals infected in the laboratory with this strain. The results suggest that media may be formulated in a similar fashion for detecting other specific strains of E. coli.     

Enterohemorrhagic Escherichia coli O157:H7 gal mutants are sensitive to bacteriophage P1 and defective in intestinal colonization

Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal(+) strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal(+) parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal(+) revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.     

Production of attaching-effacing lesions in ligated large intestine loops of 6-month-old sheep by Escherichia coli O157:1H7

Shiga-toxigenic Escherichia coli O157:H7 (STEC O157:H7) is associated with potentially fatal human disease, and a persistent reservoir of the organism is present in some farm animal species, especially cattle and sheep. The mechanisms of persistent colonisation of the ruminant intestine by STEC O157:H7 are poorly understood but may be associated with intimate adherence to eukaryotic cells. Intimate adherence, as evidenced by induction of attaching-effacing (AE) lesions by STEC O157, has been observed in 6-day-old conventional lambs after deliberate oral infection but not in older animals. Thus, the present study used a ligated intestinal loop technique to investigate whether STEC O157:H7 and other attaching-effacing E. coli may adhere intimately to the sheep large intestinal mucosa. To do this, four STEC O157:H7 strains, one STEC O26:K60:H11 and one Shiga toxin-negative E. coli O157:H7 strain, suspended in either phosphate-buffered saline or Dulbecco's modified Eagle's medium, were inoculated into ligated spiral colon loops of each of two lambs. The loops were removed 6 h after inoculation, fixed and examined by light and electron microscopy. AE lesions on the intestinal mucosa were produced by all the inoculated strains. However, the lesions were sparse and small, typically comprising bacterial cells intimately adhered to a single enterocyte, or a few adjacent enterocytes. There was little correlation between the extent of intimate adherence in this model and the bacterial cell density, pre-inoculation growth conditions of the bacteria or the strain tested.     

Adherence of Vero cytotoxin-producing Escherichia coli of serotype O157:H7 to human epithelial cells in tissue culture: role of outer membranes as bacterial adhesins

Escherichia coli of serotype O157:H7 are Vero cytotoxin-producing enteric pathogens that have recently been associated with outbreaks of haemorrhagic colitis, sporadic cases of haemorrhagic colitis and with the haemolytic uraemic syndrome. The organisms demonstrate attaching and effacing binding to the caecum and colon of orally infected gnotobiotic piglets, chickens and infant rabbits. E. coli O157:H7 cells adhere to the surface but do not invade the cytoplasm of human epithelial cell lines in tissue culture. Since outer membranes, lipopolysaccharides and flagella have been identified as bacterial adhesins on other enteric pathogens, we evaluated their roles in the binding of non-fimbriated E. coli O157:H7 to HEp-2 cells. Hyperimmune rabbit antisera were prepared to whole cells, outer membranes and flagella of E. coli O157:H7. The presence of antibody to homologous antigen was confirmed by dot blot immunoassays. Both antisera and purified outer membrane and flagellar antigens were co-incubated with bacteria and HEp-2 cells to quantitate inhibition of bacterial attachment. Adherence of E. coli O157:H7 to tissue culture cells was inhibited by rabbit antisera raised to whole cells (76.0 +/- 5.6% inhibition compared with bacterial adherence in the presence of pre-immune rabbit serum) and outer membranes (69.2 +/- 3.4% inhibition). In contrast, inhibition of bacterial attachment to tissue-culture cells was significantly less when two antisera to H7 flagella were co-incubated with E. coli O157:H7 and HEp-2 cells (12.4 +/- 7.6%; 6.0 +/- 3.5% inhibition). Outer-membrane extracts inhibited adherence to E. coli O157:H7 to HEp-2 cells in a concentration dependent manner whereas isolated flagella and lipopolysaccharide antigens did not inhibit bacterial attachment.(ABSTRACT TRUNCATED AT 250 WORDS)