DIARRHEAGENIC ESCHERICHIA COLI (E.COLI) ENTEROPATHOGENIC E.COLI (EPEC) ENTEROINVASIVE E.

COLI (EIEC)
PROFESSOR : DOCTOR MARWA MEHESSEIN ASSESSOR : DOCTOR MALAKA MOF (MICROBIOLOGY DEPARTMENT) NOR SALEHAH SAZLI (10-3-271) NUR ATHIFAH MOHAMMAD BASRI (10-3-272) NUR ADILAH FAISAL SABRI (10-3-273) NUR EZZATI AIZA MAHAT (10-3-274) NUR AQILAH TAJUDEEN (10-3-275)

Escherichia Coli
Escherichia Coli (E. Coli) is a gram negative, rod shaped bacterium. It is one of the normal floras present in intestine of human. It can turn pathogenic to human by causing food poisoning and diarrhea to human. However, most of strains of E. coli are not pathogenic to human and harmless. But there are also some strains which are pathogenic to human. They are Enteropathogenic E. coli (EPEC), Enteroinvasive E. Coli (EIEC), Enterotoxigenic E. Coli (ETEC), and Enterohaemorrhagic E. Coli (EHEC). NUR EZZATI AIZA MAHAT (10-3-274)

TYPICAL ENTEROPATHOGENIC ESCHERICHIA COLI (TYPICAL EPEC)

1. DEFINITION
Enteropathogenic Escherichia coli (EPEC) are one of the Escherichia coli that are pathogenic to human by producing diarrhea. It is characterized by action of attaching and effacing (A/E) on intestinal cells (Figure 1). It does not produce Shiga toxin, Shigalike toxin or verocytotoxin. Generally pathogenesis of EPEC characterized by microvilli destruction, close adherence of bacteria to the intestinal epithelium and also aggregation of certain cytoskeleton at the sites of bacterial attachments. It is further differentiated to typical EPC and atypical EPEC based on their genetic characteristics, serotype and virulence factors.

Figure 1 show the attaching and effacing of EPEC to the intestinal cells.

2. SEROTYPE

Majority of EPEC fall into an O:H serotypes. The serotypes can be seen in Table 1.

Table 1 shows several serotypes of typical and atypical EPEC strains.

Table 2 shows the types of intimin present in typical and atypical strains of EPEC

3. VIRULENCE
Intimin that is a virulence factor (non fimbrial adhesin). It is attaching and effacing (A/E) protein which helps in adhesion of bacteria. It is expressed by the bacteria and bind to its receptor that is translocated intimin receptor (TIR) which is present in cytoplasm of intestinal cell. Posses EAF (EPEC adherence factor) plasmid. Typical EPEC strains only show localized adherence to cultured epithelial cells which shows compact micro colonies of bacteria or bacterial clusters (Figure 2). EAF not necessarily cause A/E lesion but presence of the plasmid could enhance the efficiency of the lesion. Secondly, there is also gene to encode for the bundle forming pilli (BFP) in EAF plasmid. These sequences provide stabilization to the micro colonies of bacteria by interconnecting them together. This is shown after bacteria are in contact with the cells for 3 hours. BFP play a role in bacterial cell adhesion and somehow this could enhance the A/E lesion formation.

Figure 2 shows 4 types of adherence of Escherichia coli to the cultured epithelial cells.

Figure 3 shows different pathogenesis of different E coli.

4. PREVALENCE
Typical EPEC cause outbreaks of infantile diarrhea in developing countries. Usually typical EPEC serotype rarely cause diarrhea in industrialized countries. It is strongly related to diarrhea in child below 1 year of age. The frequency of this serotype of diarrhea of adult or children below 1 year of age was rarely seen. This condition maybe due to increased resistance, developed immunity or loss of some specific receptors for adhesion. First typical EPEC strains isolated in different countries were of serotypes O55:H6 and O111:H2. These strains also present in industrialized countries but rare. Recently, several studies showed that the emergence of typical EPEC has decreased compared to atypical EPEC. This is maybe due to development of some developing countries resulting in proper sanitation and improved hygiene.

5. RESERVOIR
Typical serotypes of EPEC are not founded in animals. So this indicates that human is the only living reservoir for this organism.

NUR AQILAH TAJUDEEN (10-3-275)

6. PATHOGENESIS
After exposure to the small intestine mucosa, the typical EPEC [tEPEC] introduce an arm-like structure called bundle-forming pilus [BFP] which promotes localized adherence. tPEC also carry the LEE (locus enterocyte & effacement) that encodes intimin and other factors responsible for initiation of attachment. Lesions produced by the tEPEC known as A/E ; attaching and effacing. The tissue related to the lesion area have characteristic morphological changes such as dissolution of intestinal wall binding surface accompanied by loss of epithelial microvilli at the site of bacteria intimately attached. According to the journal Principle Of Bacterial Pathogenesis by Eduardo A. Groisman published by Elsevier, this incident explained by the formation of cup-like projections or pedestral by the rearrangement of the cytoskeleton in host acini once the tEPEC reside. The

tEPEC also is able to move from pedestral to pedestral across the exterior of mucosal cells by utilizing compound that present at the human mucosal tissue. The interruption of tissue arrangement and molecular interaction at the site of lesions consequently lead to loss of absorptive surface area that eventually leads to diarrhea.

7. DIARRHEA CAUSED BY TYPICAL EPEC

tEPEC strains contribute to predominant case of infantile diarrhea, worldwide and represent major endemic health threat to children under 6 months of age living in developing countries In contrast to other diarrheagenic Escherichia, EPEC does not produce any classical protein toxins but induces diarrhea by intimate binding to intestinal cells. Diarrhea is the result of a series of signals triggered by the pathogen-host membrane interaction, which in turn provokes reorganization of the cytoskeleton of the affected cell, involving accumulation of polymerized actin beneath the adherent bacteria, with a consequent loss in microvillus structure and effacement of the intestinal villi. The diarrhea caused is characterized by the transient watery diarrhea, profuse watery diarrhea plus vomiting and also persistent diarrhea. Blood and mucus are rarely present in stool while the fever is either very low or absent. Noted that vomiting is very prominent in diarrhea produced by tEPEC and may render oral rehydration therapy.

8. DIAGNOSIS
Lab Diagnosis
Bacterial isolation and characterization.The swabs are streaked onto plates of MacConkey agar, and the plates incubated at 37C for 24 h. Lactose-positive and lactose-negative colonies are identified biochemically by standard techniques using EP M, MILi, and Simmons citrate. The colonies are subcultured on tryptic soy agar and also stored in brain heart infusion agar plus 50% glycerol at 70C for further utilization.

Serotyping
The O serotyping method was used to identify the isolates belonging to O serogroups defined commercially. Isolates that can agglutinate with one of the specific commercial pathogenic O antisera were defined as suspected DEC (sDEC) isolates, and they were collected for further testing to see if they carried any of the six virulence genes mentioned below. For O-antigen determination, suspend the bacterial culture in 3 ml normal saline, heated the mixture to 100C for 1 h, and used the boiled suspension as the antigenic mixture. Then mixed 1 drop of a specific O poly- or monovalent antiserum of pathogenic E. coli immune sera (Denka Seiken, Tokyo, Japan) with the antigen preparation on a glass slide for 1 min and observed the slide for agglutination. For H-antigen determination, pass the bacterial culture through the semisolid medium with a Craigie's tube to enhance the motile ability and then grew the culture in liquid broth. After the addition of a formalin solution to achieve a final concentration of 1%, the suspension could be used as an antigenic mixture and was mixed with specific H-antigen monovalent antiserum (Denka Seiken) in a plastic tube. The agglutination results could be observed after the tubes were kept in a 50C water bath for 1 h. However this serotyping is neither sensitive nor specific. Polymerase Chain Reaction (PCR) This test is responsible to differentiate and ensure the type of EPEC (typical or atypical) strains as it act on specific primers for each gene that encodes intimin, Stx-gene (Shiga- toxins) and bFp (bundle-forming pilus)

9. PREVENTION AND THERAPY

Cook all ground beef and hamburger thoroughly. Because groundbeef can turn brown before disease-causing bacteria are killed, thus try not to eat ground beef patties that are still pink in the middle.

Drink only pasteurized milk, juice, or cider. Commercial juice with an extended shelf-life that is sold at room temperature (e.g. juice in cardboard boxes, vacuum sealed juice in glass containers) has been pasteurized, although this is generally not indicated on the label. Juice concentrates are also heated sufficiently to kill pathogens. Wash fruits and vegetables thoroughly,

especially those that will not be cooked. Children under 5 years of age,

immunocompromised persons, and the elderly should avoid eating alfalfa sprouts until their safety can be assured. Methods to decontaminate alfalfa seeds and sprouts are being investigated. Drink municipal water that has been treated with chlorine or other effective disinfectants. Avoid swallowing lake or pool water while swimming. Make sure that persons with diarrhea, especially children, wash their hands carefully with soap after bowel movements to reduce the risk of spreading infection, and that persons wash hands after changing soiled diapers. Anyone with a diarrheal illness should avoid swimming in public pools or lakes, sharing baths with others, and preparing food for others.

Rehydration either by oral or parenteral solutions.

#Human milk is proved to be able to inhibit tEPEC adherent and in addition it contains antibody against several EPEC virulence factors. Studies conducted by Dr. Alfredo G.Torres, professor of the Texas University, USA among community of Latin America, shows that breast-feeding infants less than 6 months contribute to lower the risk of tEPEC infection related to diarrhea of serotype O111:H2 (non motile) and O119:H6 ##Vaccines for EPEC are still under development. Sherwold L. Gorbash stated in his bookInfectious Diseases ;( published by Lipincott, Willsons & William) that enthusiasism for an EPEC vaccine are based on the intimin, bFp or secreted proteins as these encode for the virulence and pathogenicity of the infections.

NOR SALEHAH SAZLI (10-3-271)

ATYPICAL ENTEROPATHOGENIC ESCHERICHIA COLI (ATYPICAL EPEC)

1. Serotype
Atypical EPEC is more closely related to Shiga toxin-producing E. coli (STEC), and like STEC these strains appear to be emerging pathogens. The most studied EPEC strains belong to a series of O antigenic groups known as EPEC O serogroups. Twelve EPEC serogroups were recognized by the World Health Organization in 1987: O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158. These serogroups include both typical and atypical EPEC strains, as well as other diarrheogenic E. colicategories, mainly enteroaggregative E. coli (EAEC) . Furthermore, most of the strains of each category correspond to specific serotypes in each O serogroup. The division of EPEC strains into typical and atypical has important implications that are not yet fully appreciated. EPEC can no longer be considered as a single group of enteropathogenic organisms. (1)(8) Strains Serotypes a Typical O55:H6, O86:H34, O111:[H2], O114:H2, O119:[H6], O127:H6, O142:H6, O142:H34 Atypical O26:H[11], O55:[H7], O55:H34, O86:H8, O111ac:[H8], O111:H9, O111:H25, O119:H2, O125ac:H6, O128:H2
a

Brackets denote the frequent occurrence of non motile strains.(2)

2. Prevalence
Atypical EPEC are prevalent in both developed and developing countries. They appear to cause disease in a broader range of ages and have been associated with outbreaks in developed countries.(4) The main difference between tEPEC and aEPEC is the presence of the EPEC adherence factor (EAF) plasmid in tEPEC .This plasmid encodes the bundle-forming pilus (BFP), which mediates localized adherence to intestinal cells, which is an essential property to differentiate typical EPEC from atypical EPEC strains .

3. Reservoir
For atypical EPEC, both animals and humans can be reservoirs such as dogs, cats, cattle, sheep, rabbits and monkeys. (3)
4.

Virulence Factor (2)

A subset of EPEC, known as atypical EPEC, do not carry EPEC adherence factor plasmid (pEAF) and hence do not produce bundle-forming pili (Bfp). Accordingly, their role in disease is controversial. Recently, people investigated the causes of community-acquired gastroenteritis.

Among the infectious agents that were sought in these studies was atypical EPEC, which emerged as the single most frequent pathogen in the study population. Atypical EPEC strains frequently express enteroaggregative heat stable toxin (EAST1) and other potential virulence factors not encoded in the LEE region (Table 1). Accordingly, there are two kinds of atypical EPEC strains: those that express only the LEE-encoded virulence factors and those that express both LEE and the non-LEE encoded virulence factors. Usually both kinds of strains belong to a single clone. All atypical EPEC serotypes, with exception of O125ac:H6, include both kinds of strains. All strains of this serotype examined thus far show the aggregative adherence pattern and the LEE region. The occurrence of more than one kind of strain in most atypical serotypes is another interesting difference between typical and atypical EPEC. Table 1
Virulence characteristics not encoded on the locus of enterocyte effacement (LEE) of atypical enteropathogenic Escherichia coli (EPEC) strains isolated in So Paulo, Brazil

Serotype Characteristics O26:[H11]a EAST1, E-hlyb O55:[H7] EAST1, Afa O111ac:[H8] E-hly O111:[H9] E-hly O119:H2 EAST1 O125ac:H6 AA O128:H2 EAST1
a

Brackets denote the frequent occurrence of nonmotile strains. EAST, heat-stable toxin 1 of EAEC; E-hly, EHEC hemolysin; AA, aggregative adherence; Afa, afimbrial adhesin.
b

Typical and atypical EPEC strains also differ in adherence patterns. The typical strains show only the LA pattern, while atypical strains may show the LAL (localized-like adherence) pattern , the DA (diffuse adherence) pattern, or the AA (aggregative adherence) pattern (Figure 1). The LAL pattern is characteristic of the strains of most serotypes and is mediated mainly by intimin . The DA pattern is mediated by the Afa adhesin and the AA is mediated by an aggregative adhesin. Typical and atypical EPEC also have some interesting differences with regard to the intimin types (Table 2).

Table 2
Intimin types of typical and atypical enteropathogenic Escherichia coli (EPEC) serotypes

Intimin types Alpha Beta Gamma Delta

a

Typical O55:[H6],a O127:H6, O142:H6, O142:H34 O111:[H2], O114:H2, O119:[H6]

Atypical O111:[H9], O125ac:H6 O26:H[11], O119:H2, O128:H2 O55:[H7], O111ac:[H8]

O86:H34

Brackets denote the frequent occurrence of nonmotile strains.

FIGURE 1: Adherence patterns of enteropathogenic Escherichia coli (EPEC) strains. Localized adherence (LA), diffuse adherence (DA), aggregative adherence (AA), and localized adherencelike (LAL). Magnification: X100.

5. Pathogenicity associated and diarrhea

Atypical EPEC seems to be a more important cause of diarrhea. Patients infected with atypical EPRC experienced mild, nondehydrating and non inflammatory diarrhea that was not particularly associated with fever, vomitting or abdominal pain. However, the duration of diarrhea in patients infected with atypical EPEC was significantly longer than that caused by other species or where no pathogens were identified. Infection with atypical EPEC is associated with prolonged diarrhea.(6)

6. Diagnosis
SOURCES / SPECIMENS: Stools and fecally contaminated material Stool culture is a common method used to identify E. coli. DNA probes and techniques such as PCR can be applied directly to clinical samples and food. Both typical and atypical EPEC are most frequently identified by detection of the eae gene encoding the intimin protein. The presence of the eae gene and demonstration of the absence of the verotoxin (enterotoxin) gene are absolutely required for the molecular identification of EPEC .(7) Serotyping of atypical strains is insufficient to assess the pathogenic properties of such strains, because such organisms are quite variable in their repertoire of virulence determinants. We have applied genetic and phenotypic analysis to a collection of 118 typical and atypical strains of EPEC and non-EPEC serogroups, to identify common and unique virulence loci and traits in these organisms. We determined the presence of and some characteristics of the LEE region, and we searched for the occurrence of virulence-associated markers within the E. coli species. Furthermore, we also determined their adherence patterns and serotypes. These data can be used to detect atypical EPEC in clinical specimens and to elucidate the role of specific virulence factors.(5)

7. Special treatment and vaccine

Treatment with trimethoprim/sulfamethoxazole (TMP-SMX) or quinolones reduces the duration of diarrhea .Treatment of fluid and electrolyte loss is usually achieved through oral rehydration. The use of the World Health Organization Oral Rehydration Salts (ORS) solution has been recommended. Intravenous rehydration may be necessary for infants, individuals with excessive vomiting, or those with severe dehydration. Bismuth subsalicylate may decrease the amount of diarrhea and the duration of disease. Antimicrobial therapy is generally not indicated, because of the self-limited nature of most of these diseases.

IMMUNIZATION: There are currently no vaccines approved for human use against diarrheagenic E. coli.

NUR ADILAH FAISAL SABRI (10-3-273)

ENTEROINVASIVE ESCHERICHIA COLI (EIEC)

Escherichia coli is a gram negative bacteria that normally inhabit the intestine of humans and animals. There are strains that are harmless and some are pathogenic to humans. For instance, EIEC. These organisms are pathogenically so closely related to Shigella species. This bacterium is classified as one of the diarrheogenic E.Coli.

The picture above showing Enteroinvasive E.coli Source : blog.naver.com

1. DEFINITION
The strains of Escherichia Coli that invade or penetrates the gut mucosa and multiplies in the colon epithelial cells, resulting Shigellosis like changes of the mucosa. This strain will produce severe diarrhea illness that can resemble shigellosis expect for the absence of vomit, shorter duration of illness and number of bowel movements.

2. SEROTYPES
There are about 243 serotypes that were studied and isolated in different regions of the country over the period 1954-1988 and at least 106 organisms can cause illness. The strain that are known as enteroinvasive E.Coli (EIEC) belong to the following O serotypes: O28ac:NM; O29:NM; O112ac:NM; O121:NM; O124:NM; O124:H30; O164:NM; O167:NM. EIEC 0124 is the most frequently isolated and is associated with sporadic cases in travellers. Occasional outbreaks related to ingestion of contaminated water or food and from person to person transmission of the disease. EIEC 0164 is the most prevalent serotype that is isolated in Bulgaria.

3. PREVALENCE
Enteroinvasive Escherichia Coli was first isolated in Italy during the World War II. This bacterium can cause dysentery but it is less reported than other etiological agents that can cause diarrhoea all over the world. Any age of people all over the world are susceptible to be infected by this bacterium, but can cause severe manifestations in very young and very old persons. It is most commonly isolated in developing countries and in rural and slum areas. This is due to poor sanitation and low socioeconomic status. It is fewer in developed countries and in cities. Outbreaks of food-borne infections due to EIEC have also been reported elsewhere, mostly from unpasteurized milk and uncooked hamburger.

4. RESERVOIR
There is no specific known animal reservoir of EIEC. The primary source for EIEC is human. Evidence suggested that illness is transmitted by contamination of food and water and from person to person especially by food handlers.

Diagram above showing uncooked burger is a source of E.coli Source : www.doctortipster.com

5. VIRULENCE FACTORS:
Virulence literally means the degree of pathogenicity of a group organisms indicated by the ability of the organisms to invade tissues. Some of the methods are by adhesions, invasions and produce toxins. - Similarly to Shigella, EIEC has the ability to invade colonic epithelium and multiplies in the cell causing destruction but it is apparently lack of fimbrial adhesins but do possesses adhesins (an outer membrane protein) to interact with the epithelial mucosa. Unlike Shigella, they do not produce LT or ST toxins. - EIEC also secretes Ipas (Invasion plasmid antigens) A-D into a host cell and trigger several events that will lead to membrane ruffling, phagocytosis and bacterial engulfment. Once internalized, bacteria is surrounded by an inclusion membrane in the cytoplasm of the host cell. -A system of Type III secretion genes are important to modify the host cell signalling and membrane lysis are encoded in their plasmid. They possesses a plasmid that encodes an outer membrane protein called IcsA that is located at one pole of the bacterial cell which propels through the bacterial cytoplasm like head of comet tail. This will trigger actin polymerization

that is necessary for spread of the bacteria to another host cell by propelling outward and pushing into the adjacent cell but not to the bloodstream therefore avoiding extracellular response. - The conclusion is, this bacteria can interact with the host cell invade by endocytosis lysis of the endocytotic vacuole bacterial multiplication spread to adjacent cells. The virulence factors for these steps are encoded on a 140 MDA plasmid. Defect of this plasmid make it non-pathogenic. SERENY Test is used to test the invasiveness of an organism including EIEC. This test is done by inoculating the bacteria into pigs eye and is positive if it results in mucopurulent and keratoconjunctivitis.

Diagram above showing the mechanism of invasion of Enteroinvasive Escherichia Coli. Source : www.nature.com

NUR ATHIFAH BINTI MOHAMMAD BASRI (10-3-272)

6. PATHOGENESIS
EIEC closely resemble Shigella in their pathogenic mechanisms. Although the infective dose of Shigella is low (in the range of 10 to few hundred cells), volunteer feeding studies showed that at least 106 EIEC organisms are required to cause illness in healthy adults. EIEC penetrate and multiply within epithelial cells of the colon causing widespread cell destruction. EIEC invade the epithelium from the intestinal lumen through M-cells. After reaching the epithelium, they invade epithelial cells and are phagocytosed by resident macrophages. EIEC escape the phagosome of both cells but while EIEC replicate within epithelial cells, they induce apoptosis in macrophages. Bacteria are released and can invade the epithelial cells from the basolateral side, move into the cytoplasm by triggering actin polymerization, and spread to adjacent cells.

Figure 1: EIEC infection of colonic epithelial cells Reference:http://www.hindawi.com/journals/bmri/2013/374395/fig1/

7. DIAGNOSIS
During the early (acute) phase of infection, large numbers of EIEC are excreted in the faeces. Contaminated feces samples can be examined in the laboratory for diagnosis. These EIEC strains of E. coli can be distinguished from the many other E. coli in the feces by a number of special tests (including immunochemical, tissue culture, and gene probe tests). The lab diagnosis of EIEC takes about 3 days. Below are several tests that can be done to diagnose: 1. PCR : Scientist has developed multiplex PCR assays that detect EIEC isolates. The targets selected for EIEC isolates (and also Shigella spp) are ipaH. The ipaH sequences are present at multiple sites on both the large invasive plasmid and the chromosomes in EIEC strains and also in Shigella spp. These PCR were specific and sensitive for rapid detection of target isolates in stools. 2. Serotype marker: To correctly identify diarrheagenic E.coli strains, these organisms must be differentiated from non-pathogenic members of the normal flora. Serotype markers correlate, sometimes

very closely, with specific categories of E.coli ; however, these marker are rarely sufficient in and of themselves to reliably identify a strain as diarrheagenic. 3. HEp-2 cell adherence and DNA hybridization: Due to insufficiency of serotype marker to identify diarrheagenic strain of E.coli, the detection of it has focus increasingly on the identification of certain characteristics which themselves determine the virulence of these organisms which include Hep-2 cell adherence and DNA hybridization.

8. TREATMENT
Treatment of EIEC infection is dependent on the severity of the symptoms, the age of the patients, and coo morbidities (such as diabetes etc). Treatments include: 1. Avoidance of dehydration and rehydration i. Oral therapy - if vomiting and dehydration are not severe. Small amounts and often, ideally with and balanced electrolyte solutions, but other fluids can be used. Avoid high sugar drinks as this may worsen diarrhea and dehydration. ii. Nasogastric therapy - in a hospital setting may be used to avoid intravenous therapy. iii. Intravenous therapy - where vomiting and/or dehydration are severe, or there is an altered level of consciousness or other coo morbidities. 2. Treatment of other symptoms i. Pain and fever can be treated with paracetamol or ibuprofen ii. Anti-emetics - can be useful where vomiting is a predominant feature, but generally not recommended in children. iii. Antidiarrheals - should be avoided because of the risk of bacteremia. 3. Antibiotics such as TMP-SMX ( trimethoprim-sulfamethoxazole) It helps to eradicate susceptible strains of EIEC (that are not resistant) from intestines. 4. Hospitalisation. Recommended for: i. The very young (<6 months) and the very elderly ii. Moderate to severe dehydration and ongoing losses iii. Those with other significant medical conditions iv. Altered level of consciousness

5. Refeeding i. Early age appropriate refeeding is now recommended once vomiting is controlled and rehydration is complete ii. Use complex carbohydrates such as rice, potatoes, and bread; and lean meats iii. Delay in reintroduction of non-human milk has previously been recommended due to the risk of lactose intolerance, but there is an increasing body of evidence that suggests reintroduction of milk once tolerated, or even continuing milk during an acute illness, is not associated with increased adverse outcomes iv. Breastfeeding should continue as tolerated 6. Public Health measures and good hygiene i. To avoid spread of disease ii. To identify the source of the disease

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NUR EZZATI AIZA BINTI MAHAT

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NUR AQILAH TAJUDEEN

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NOR SALEHAH SAZLI

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