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Lesson N4: LABORATORY DIAGNOSIS OF ESCHERICHIOSIS1.Scientifically methodical ground of theme Strains of Escherichia coli a...
widely spread serotypes is designated 0111 : K58 : H2. Under the effect of transformation, lysogenic conversion,transducti...
The corresponding 0-group to which an enteropathogenic-serovars belong is determined by means of theagglutination reaction...
Like CT, LT causes diarrhea by stimulating the activity of a membrane-bound adenylate cyclase.This resultsin the conversio...
are lysogenic for one or more bacteriophages that encode for the production of one or both of two antigenicallydistinct to...
The most recent of these has been termed the enteroaggregative E. coli. These strains seem to cause diarrheathrough their ...
7. Methods of diagnosis of colienterites and other diseases, which caused by E. coli. Peculiarities of gettingof tested ma...
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Bohomolets Microbiology Lesson #4


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  1. 1. Lesson N4: LABORATORY DIAGNOSIS OF ESCHERICHIOSIS1.Scientifically methodical ground of theme Strains of Escherichia coli and related Gram-negative bacteria predominate among the aerobic commensal florapresent in the gut of man and animals. Individuals have a rich flora in the lower ileum and, more specially, in thecolon, which includes Escherichia coli. It is acquired by ingestion of the organisms during the first few days afterbirth. The species Escherichia coli contains a great variety of strains that include purely commensal organisms aswell as those possesing combinations of virulance determinants that enable them to act as spesific pathogens of thegut and extraintestinal sites, especially the urinary tract.2.Educational purpose Students must know: 1. Structure, staining properties ànd cultivation E. coli. 2. Antigenic structure of E. coli, their classification. 3. Fermentative properties and toxin production of E. coli 4. Main clinical forms of escherichiosis. 5. Methods of laboratory diagnosis of diseases, main methods of prophylaxis and treatment. Students should be able to: – prepare the smears from pure culture; – stain the smears by Gram’s technique; – make microscopical examination of the smears; - carry out agglutination test for determination of serovar of E. coli. 3.Chart of topic content. Escherichia coli. The organism was isolated from faeces in 1885 by T. Escherich.E.coli is a commoninhabitant of the large intestine of humans and mammals. It is also found in the guts of birds, reptiles, amphibians,and insects. The bacteria are excreted in great numbers with the faeces and are always present in the externalenvironment (soil, water, foodstuffs, and other objects). Morphology. E coli are straight rods measuring 0.4-0.7 in breadth and 1-3 in length. They occur asindividual organisms or in pairs and are marked by polymorphism. There are motile and non-motile.The cellsurface has pili on which certain phages are adsorbed. The microcapsule is not always clearly defined. Cultivation. E. coli is a facultative anaerobe. The optimum temperature for growth is 30-37 °C and theoptimum pH value of medium up 7.2-7.5. The organism also grows readily on ordinary media at room temperatureand at 10 and 45 °C, growth becomes visible in the first two days. E. coli from cold-blooded animals grows at22-37° C but not at 42-43° C. On meat-peptone agar E. coli produces slightly convex semitransparent, greyish colonies, and in meat brothit forms diffuse turbidity and a precipitate. The organism produces colonies which are red on Ploskirevs medium,red with a metallic hue on Endos medium, and dark-blue on Levins medium. Fermentative properties.Glucose, lactose, mannitol, maltose and many other sugers are fermented withproduction of acid and gas. Tipical strains do not fermant sucrose. Gelatin is not liquified, H2S is not formed, irea isnot split and growth does not occur in KCN medium. Virulence factors. Certain strains of E. coli are conditionally pathogenic. They contain a gluco-lipo-proteincomplex with which their toxic, antigenic, and immunogenic properties are associated. Some strains possesshaemolytic properties (O124 and others) determined by plasmids. Pathogenic cultures possess endotoxins andthermolabile neurotropic exotoxins. The latter accumulate in broth cultures on the second-fourth day of cultivation,while the endotoxins appear only after the twentieth day. Haemotoxins and pyrogenic substances, proteinases,deoxyribonucleases, urease, phosphatase, hyaluronidase, amino acid decarboxylases have been obtained frompathogenic strains. Antigenic structure. The antigenic structure of E. coli is characterized by variability and markedindividuality. Along with the H- and O-antigens, the presence of other antigens has been shown m some strains, i.e.the surface somatic (membranous, capsular) K-antigens which contain the thermolabile L- and B-antigens and thethermostable A- and M-antigens. Each antigen group in its turn is composed of a number of antigens designated by Arabic numbers, e.g. theO-group has 173 antigens, the K-subgroup 90, the H-subgroup 50, etc. On the basis of antigenic structure anantigenic formula is derived which fully reflects the antigenic properties of the strain.For example, one of the most 1
  2. 2. widely spread serotypes is designated 0111 : K58 : H2. Under the effect of transformation, lysogenic conversion,transduction, and conjugation E. coli may change its antigenic properties. Numerous varieties of the organism are produced on cultivation under artificial conditions. Such varieties arenot only of theoretical interest, but also of great practical importance in laboratory diagnosis of enteric infections. Classification. The genus Escherichia includes E. coli, E. freundi, E. intermedia, and others. E. coli speciesconsisting of several biotypes and serotypes. They are differentiated according to cultural, biochemical, andserological properties F. Kauffmann has detected 25 O-groups responsible for various diseases in humans. About 50 phage variants have been revealed among E. coli organisms. They are used in laboratory diagnosisas confirmatory characteristics of the isolated serotypes. Resistance. E. coli survives in the external environment for months. It is more resistant to physical andchemical factors of the external environment than the typhoid and dysentery bacteria. E. coli is killedcomparatively rapidly by all methods and preparations used for disinfection. At 55° C the organism perishes in 1hour, and at 60° C in 15 minutes. E. coli is sensitive to brilliant green. E. coli is used as a test microbe in the assay of disinfectants and methods of disinfection and also in titrationof certain antibiotics. Pathogenesis and diseases in man. Definite E. coli serogroups are capable of causing various acuteintestinal diseases in humans: (1) the causative agents of colienteritis in children are O-groups-25, -26, -44, -55,-86, -91, -111, -114, -119, -125, -126, -127, -128, -141, -146, and others (they cause diseases in infants of the firstmonths of life and in older infants); (2) the causative agents of dysentery-like diseases are E. coli of the O-groups-23, -32, -115, -124, -136, -143, -144, -151, and others; (3) the causative agents of cholera-like diarrhoea arethe O-groups-6, -15, -78, -148, and others, they produce thermolabile and thermoresistant enterotoxins. Colienteritis begins acutely with high temperature (38-39 °C), and frequently with severe meteorism,vomiting, diarrhoea, and general toxicosis. The disease usually occurs in infants of the first year of life. The infection is acquired from sick children or carriers. Pathogenic E. coli serovars are found on variousobjects. It is assumed that colienteritis is transmitted not only by the normal route for enteric infections but alsothrough the respiratory tract by the droplets and dust. The pathogenesis of colienteritis depends entirely on the organisms condition. In prematurely born infantsand in infants during the first months of life the bactericidal activity of blood is considerably lower in respect to thepathogenic E. coli serovars in comparison to the nonpathogenic types. The reactivity of the childs body at the timeof infection plays an important role in the mechanism of resistance to the pathogenic strains. The pathologicalprocess develops mainly in the small intestine. Most probably, the mucous membrane of the small intestine inparticular is exposed to the action of thermolabile toxic substances. Serovars O-124, O-151 and others causediseases which are similar to dysentery. E. coli may cause colibacillosis in adults (peritonitis, meningitis, enteritis, toxinfections, cystitis, pyelitis,pyelonephntis, angiocholitis, salpingooophontis, appendicitis, otitis, puerperal sepsis, etc.). Overstrain, exhaustion,and conditions following infectious diseases facilitate the onset of various E. coli infections. In a number of casesthe organism is responsible for food poisoning. Immunity. In individuals who had suffered from diseases caused by pathogenic E. coli serovars, crossimmunity is not produced owing to which reinfection may occur. Over 85 per cent of E. coli strains containinhibiting substances, colicins, marked by antagonistic properties in relation to pathogenic microbes of the entericgroup, they are used as therapeutic and preventive agents, e.g. colibacterin (E. coli M 17, etc.). Besides this, E coli as well as other common inhabitants of the intestine are capable of synthesizing variousvitamins (K2, E, and group B) which are indispensable to the human organism. The ability of various E. coliserovars to suppress the growth of Mycobacterium tuberculosis has also been observed. The suppression of E. coliand other members of the biocoenosis may result in a chronic disease known as dysbacteriosis. Laboratory diagnosis. The patients faeces, throat and nasal discharges, material obtained at autopsy(blood, bile, liver, spleen, lungs, contents of the small and large intestine, pus), water, foodstuffs, and samples ofwashings from objects and hands of staff of maternity hospitals, hospitals, and dairy kitchens are all used forlaboratory examination during colienteritis. If possible, faecal material should be inoculated immediately after ithas been collected. The throat and nasal discharges are collected with a sterile swab. Specimens of organs obtainedat autopsy are placed in separate sterile jars. The tested material is inoculated onto solid nutrient media (Endos, Levins) and, simultaneously, ontoPloskirevs media and bismuth-sulphite agar for isolation of bacteria of the typhoid-paratyphoid and dysenterygroup.Blood is first inoculated into broth and then subcultured on solid media when development of a septicprocess is suspected. Pus is collected for examination in suppurative lesions. It is placed into a dry sterile vesseland then inoculated onto the differential media of Endo or Levin. The pure culture isolate is identified by itsmorphological, cultural, biochemical, serological, and biological properties. 2
  3. 3. The corresponding 0-group to which an enteropathogenic-serovars belong is determined by means of theagglutination reaction after the K-antigen of the culture that is being studied has been destroyed by boiling. Besides, the immunofluorescence method employing type specific labelled sera is also used. It yields apreliminary answer in one to two hours. In serological diagnosis of colienteritis beginning with the third to fifth day of the disease the indirecthaemagglutination reaction is used which excels the agglutination reaction in sensitivity. It is positive when theantibody titre grows in the course of the diseased. Treatment. Patients with colienteritis are prescribed antibiotics (tetracycline with vitamins C, B 1 and B2)and biopreparations (coli autovaccine, coli bacteriophage, colicin, bacterin, lactobacterin, bificol, bifidumbacterin).Physiological solutions with glucose are injected for controlling toxicosis. Prophylaxis. To prevent diseases caused by pathogenic serovars of E. coli, special attention is given toearly identification of individuals suffering from colienteritis, and also to their hospitalization and effectivetreatment. Regular examination of personnel is necessary in childrens institutions as well as of mothers whosechildren are suffering from dyspepsia. Considerable importance is assigned to observation of sanitary regulations inchildrens institutions, infant-feeding centres, maternity hospitals, and nurseries. Protection of water and foodstuffsfrom contamination with faeces, the control of flies, and gradual improvement of standards of hygiene of thepopulation are also particularly important. Sanitary significance of E. coli. This organism is widely spread in nature. It occurs in soil, water,foodstuffs, and on various objects. For this reason E. coli serves as an indicator of faecal contamination of theexternal environment. Detection of E. coli is of great importance in estimating the sanitary index of faecal contamination of water,foodstuffs, soil, beverages, objects, and hand-washings. The degree of contamination of water, soil and foodstuffsis determined by the coli titre or coli index.Faecal contamination of articles of use is estimated by qualitativedetermination of the presence of E. coli. Pathogenicity of Escherichia coli. Although E. coli is part of the normal flora of the intestinal tract, it isalso the most common gram-negative pathogen responsible for nosocomially acquired septic shock, meningitis inneonates, cystitis and pyelonephritis in women, and for several distinct forms of diarrheal disease and dysenteryaffecting populations throughout the world. Strains of E coli capable of causing such diseases possess one or morevirulence factors that are not found in E. coli strains comprising the normal flora. Such virulence factors can becharacterized as follows, the capacity to adhere to specific mammalian cells; the ability to invade and growintracellularly in intestinal epithelial cells; the secretion of one or more enterotoxins that cause fluid loss, resultingdiarrhea; the formation of a cytotoxin that blocks protein synthesis, causing a hemorrhagic colitis; and thepossession of an antiphagocytic capsule that is responsible, at least in part, for the bacteremia and meningitiscaused by E. coli. In addition, the ability to obtain iron from transferrin or lactoferrin by the synthesis of iron-binding siderophores markedly enhances the virulence of such strains through their ability to grow in host tissues.No one strain of E. coli possesses all of these properties but all pathogenic strains must have one or more virulencefactors to produce disease. Diarrheal Diseases. Diarrhea kills more people worldwide than AIDS and cancer, with about five milliondiarrheal deaths occurring annually primarily because of dehydration. Most of these occur in neonates and youngchildren, anda large number are caused by pathogenic E. coli. The disease in adults, known by many names such astraveller’s diarrhea or Montezumas revenge, may vary from a milddisease with several days of loose stools to asevere and fatal cholera-like disease. Such life-threatening E. coli infections occur throughout the world but aremost common in developing nations. The virulence factors responsible for diarrheal diseaseare frequently encoded in plasmids, which may bespread from one strain to another either through transduction : or by recombination. As a result, variouscombinations of virulence factors have occurred, which has been used to place the diarrhea-producing strains of E.coli into various groups based on the mechanism of disease production Enterotoxigenic Escherichia coli. Enterotoxin-producing E coli, called enterotoxigenic E.coli (ETEC),produce one or both of two different toxins – a heat labile toxin called LT and a heat-stable toxin called ST. Thegenetic ability to produce both LT and ST is controlled by DNA residing in transmissible plasmids called entplasmids. Both genes have been cloned, and the ST gene has been shown to possess the characteristics of atransposon. HEAT-LABILE TOXIN. The heat-labile toxin LT, which is destroyed by heating at 65 °C for 30 minutes,has been extensively purified, and its mode of action is identical to that described for cholera toxin (CT). LT has amolecula weight of about 86,000 daltons and is composed of two subunits, A and B Subunit A consists of onemoleculeof Ai (24,000 daltons) and one molecule of A2 (5000daltons) linked by a disulfide bridge. Each A unit isjoined noncovalently to five B subunits. 3
  4. 4. Like CT, LT causes diarrhea by stimulating the activity of a membrane-bound adenylate cyclase.This resultsin the conversion of ATP to cyclic AMP (cAMP). cAMP induce the active secretion of Cl– and inhibit the absorption of NaCI, creating an electrolyte imbalanceacross the intestinal mucosa, resulting in the loss of copious quantities of fluid and elec-trolytes from the intestine. The mechanism by which LT stimulates the activity of the adenylate cyclase is as follows: (1) The B subunitof the toxin binds to a specific cell receptor, GM1 ganglioside, (2) the A1 subunit is released from the toxin andenters the cell; and (3) the A1 subunit cleaves nicotinamide-adenic dinucleonde (NAD) into nicotinamide and ADP-ribose and, together with a cellular ADP-ribosylating factor, transfers the ADP-ribose to aGTP-binding protein.The ADP-ribosylation of the GTP-binding protein inhibits a GTPase activity of the binding protein, leading toincreased stability of the catalytic cornplex responsible for adenylate cyclase activity. This results in an amplifiedactivity of the cyclase and a corresponding increase in the amount of cAMP produced. Two antigenically distinct heat labile toxins are produced by various strains of E. coli. LT-I is structurallyandantigenically related to CT to an extent that anti-CT will neutralize LT I LT-II has, on rare occasions, beenisolated from the feces of humans with diarrhea, but it is most frequently isolated from feces of water buftalos andcows LT-II is biologically similar to LT-I, but it is notneutralized by either anti-LT-I or anti-CT. LT will bind to many types of mammalian cells, and its ability to stimulate adenylate cyclase can be assayedincell cultures. A report has also shown that CT stimulated an increase in prostaglandin E (PGE), and that PGE1 and PGE2caused a marked fluid accumulation in the ligated lumen of rabbit intestinal segments. The mechanism whereby CTinduces PGE release is unknown. HEAT-STABLE TOXIN The heat-stable toxin STa consists of a family of small, heterogeneouspolypeptides of 1500 to 2000 daltons that are not destroyed by heating at 100 °Cfor 30 minutes. STa has no effecton the concentration of cAMP, but it does cause a marked increase the cellular levels of cyclic GMP (cGMP).cGMP causes an inhibition of the cotransport of NaCI across the intestinal wall, suggesting that the action of STamay be primarily antiabsorptive compared with that of LT, which is both antiabsorptive and secretory. STa stimulates guanylate cyclase only in intestinal cells, indicating that such cells possess a uniquereceptorfor Sta. The cell receptor for STa is known to be either tightly coupled to, or a part of, a particulate form ofguanylate cyclase located in the brush border membranes of intestinal mucosal cells. Also, intimately associatedwith this complex is a cGMP-dependent protein kinase that phosphorylates a 25,000 dalton protein in the brushborder. It has been proposed that this phosphorylated protein might be the actual mediator for the toxin-inducediontransport alterations that lead to fluid loss. The usual assay for STa is to inject the toxin intragastrically into a 1– to 4-day old suckling mouse and measure intestinal fluid accumulation (as a ratio of intestinal/remaining bodyweight) after 4 hours. STa may also be assayed directly by measuring its effect on the increase in guanylatecyclasein homogenized intestinal epithelial cells. A second heat stable toxin that is produced by somestrains of E. coli has been termed STb. This toxin isinactivein suckling mice but will produce diarrhea in weaned piglets. STb producers have not been isolated fromhumans. It does not seem to increase the level of adenylate or guanylate cyclase in intestinal mucosal cells, butmaystimulate the synthesis of prostaglandin E2. The end resuit is to enhance net bicarbonate ion secretion. COLONIZATION FACTORS. Animals also are subjects to infections by their own strains of ETEC, andsuch infections in newborn animals may result in death from the loss of fluids and electrolytes. Extensive studies ofstrains infecting newborn calves and piglets (as well as humans) have revealed that, in addition to producing anenterotoxin, such strains possess one of several fimbriate surface structures that specifically adhere to the epithelialcellslining the small intestine. These antigens (K-88 for swine strains, and K-99 for cattle) usually are fimbriatestructures that cause the toxin-producing organisms to adhere to and colonize the small intestine. The need for thiscolonizing ability is supported by the fact that antibodies directed against the colonizing fimbriae are protective. Analogous human ETEC strains also possess fimbriate structures that have been designated ascolonizationfactors (CFA). At least five such serologicallydifferent factors, CFA/I, CFA/II, CFA/III, E8775,andCFA/V, have been described. Interestingly, these colonization factors also are plasmid mediated, and singleplasmids have been described that carry genes for both CFA/I and STa. Enterohemorrhagic Escherichia coli. The enterohemorrhagic E. coli (EHEC) were first described in 1982when they were shown to be the etiologic agent of hemorrhagic colitis, a disease characterized by severe abdominalcramps and a copious, bloody diarrhea. These organisms are also known to cause a condition termed hemolytic-uremic syndrome (HUS), which is manifested by a hemolytic anemia, thrombocytopenia (decrease in the numberof blood platelets), and acuterenal failure. HUS occurs most frequently in children. Although most initially recognized EHEC belong to serotype O157:H7, other EHEC serotypes such asO26, O111, O128, and O143 have been recognized. These organisms are not invasive, but they do possess a 60-megadalton plasmid that encodes for a fimbrial antigen that adheres to intestinal epithelium. In addition, the EHEC 4
  5. 5. are lysogenic for one or more bacteriophages that encode for the production of one or both of two antigenicallydistinct toxins. These toxins are biologically identical and antigenically similar to the toxins formed by Shigelladysenteriae (Shigas bacillus), and are designated as Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT II).Because the Shiga-like toxins initially were characterized by their ability to kill Vero cells, a cell line developedfrom African green monkey kidney cells, they also arecalled Verotoxin I and Verotoxin II. SLT I consists of an A subunit and five B subunits. The sequence of the B subunit from S. dysenteriae type 1is identical to that of the B subunit of SLT I. The B subunit binds specifically to a glycolipid in microvillusmembranes, and the released A subunit stops protein synthesis by inactivating the 60S ribosomal subunit. Thisinactivation results from the N-glycosidase activity of the toxin, which cleaves off an adenine molecule (A-4324)from the 28S ribosomal RNA, causing a structural modification of the 60S subunit, resulting in a reduced affinityfor EF-1 and, thus, an inhibition of aminoacyl- tRNA binding. The consequence of toxin action is a cessation ofprotein synthesis, the sloughing off of dead cells, anda bloody diarrhea. Notice that SLT 1 carries out the samereaction as the plant toxins ricin and abrin. SLT II is biologically similar to SLT I, but because only a 50% to 60% homology exists between the twotoxins, it is not surprising that they are antigenically distinct. Interestingly, both STL I and STL-II can betransferred to nontoxin producing strains of E. coli by transduction. Outbreaks of hemorrhagic colitis have been traced to contaminated food as well as to person to persontransmission in nursing homes and day care centres. Contaminated, undercooked hamburger meat seems to be themost frequently implicated source of food borne illnesses followed by contaminated milk and water, indicatingthatcattle are a common reservoir for EHEC. Of note is that E. coli 0157:H7 has been shown to survive up to 9months at -20°C in ground beef. Thus, the EHEC are able to cause hemorrhagic colitis as a result of their ability to adhere to the intestinalmucosa, and they presumably destroy the intestinal epithelial lining through their secretion of Shiga like toxins.The mechanism whereby the EHEC cause HUS is unclear but seems to follow bloodstream carriage of SLT II tothe kidney. Experimental results have shown that humanrenal endothelial cells contain high levels of receptor forSLT-2. Moreover, in the presence of interleukin (IL)1/β, the amount of receptor increases, enhancing theinternalization of the toxin and the death of the cell. The section, "A Closer Look," describes several epidemics of hemorrhagic colitis that have occurred in theUnited States and techniques that are used for the identification of this serotype Enteroinvasive Escherichia coli The disease produced by the enteroinvasive E. coli (EIEC) is indistinguishable from the dysentery producedby members of the genus Shigella, although the shigellae seem to be more virulent.The key virulence factorrequired by the EIEC is the ability to invade the epithelial cells. EIEC INVASION. The specific property that provides these organisms with their invasive potential is farfrom understood. It is known, however, that this ability is encoded in a plasmid and that the loss of the plasmidresults in a loss of invasive ability and a loss of virulence. Moreover, the shigellae seem to possess the sameplasmid, because Western blots show that shigellae and EIEC plasmids express polypeptides that are similar inmolecular weight and antigenicity. Enteropathogenic Escherichia coli. The enteropathogenic E. coli (EPEC) are diffusely adherent organismsthat are particularly important in infant diarrhea occurring in developing countries, where they may cause amortality rate as high as a 50%. They comprise a mixture of organisms that seem to produce diarrhea by a two-stepprocess. The classic EPEC exist among a dozen or so different serotypes, all of which are characterized by thepossession of a 55 to 65-megadalton plasmid that encodes for an adhesin termed EPEC adherence factor (EAF).EAF causes a localized adherence of the bacteria to enterocytes of the small bowel, resulting in distinctmicrocolonies. This is followed by the formation of unique pedestal-like structures bearing the adherent bacteria.These structures have been termed attaching and effacing lesions. The ability to form the effacing lesion resides inan attaching and effacing gene (eae). The lesions are characterized by a loss of microvilli and a rearrangement ofthe cytoskeleton, with a proliferation of filamentous actin beneath are as of bacterial attachment. Thus, the ability of the EPEC to cause diarrhea involves two distinct genes, EAF and eae. The end result isanelevated intracellular Ca+2 level in the intestinal epithelialcells and the initiation of signal transduction, leading toprotein tyrosine phosphorylation of at least two eucaryotic proteins. EPEC strains routinely have been considered noninvasive, but data have indicated that such strains caninvadeepithelial cells in culture. However, EPEC strains do not typically cause a bloody diarrhea, and thesignificance of cell invasion during infection remains uncertain. Other Diairhea-Producing Escherichia coli. All possible combinations, deletions, or additions of thevarious virulence factors responsible for intestinal fluid loss result in diarrhea producing strains that do not fit thecategories already described. Such has been found tobe the case. 5
  6. 6. The most recent of these has been termed the enteroaggregative E. coli. These strains seem to cause diarrheathrough their ability to adhere to the intestinal mucosa and possibly by yet a new type of enterotoxin. It seemspossible that the acquisition of other virulence factors may result in the discovery of additional pathogenic strainsof E. coli. E. coli Urinary Tract Infections. Escherichia coli is the most common cause of urinary tract infections ofthe bladder (cystitis) and, less frequently, of the kidney (pyelonephritis). In either case, infections usually are of anascending type (enter the bladder from the urethra and enter the kidneys from the bladder). Many infections occurin young female patients, in persons with urinary tract obstructions, and in persons requiring urinary catheters, andthey occur frequently in otherwise healthy women. Interestingly, good data support the postulation that certainserotypes of E. coli are more likely to cause pyelonephritis than others. Thus, the ability to produce P-fimbriae (socalled because of their ability to bind to P blood group antigen) has been correlated with the ability to produceurinary tract infections, seemingly by mediating the adherence of the organisms to human uroepithelial cells. Ofnote is that the rate of nosocomial urinary tract infection per person-day was significantly greater in patients withdiarrhea, particularly in those with an indwelling urinary catheter. In addition to fimbrial adhesins, a series of afimbrial adhesins has been reported. Their role in disease is notyet firmly established, but it has been demonstrated that at least one afimbrial adhesins mediated specific binding touroepithelial cells. Recurrent urinary tract infections in premenopausal, sexually active women frequently can be prevented bythe postcoital administration of a single tablet of an antibacterial agent such as trimethoprim-sulfamethoxazole,cinoxacin, or cephalexin. E. coli Systemic Infections. About 300,000 patients in United States hospitals develop gram-negativebacteremia annually, and about 100,000 of these persons the of septic shock. As might be guessed, E. coli is themost common organism involved in such infections. The ultimate cause of death in these cases is an endotoxin-induced synthesis and release of tumor necrosis factor-alpha and IL-1, resulting in irreversible shock. The newborn is particularly susceptible to meningitis, especially during the first month of life. A survey of132 cases of neonatal meningitis occurring in the Netherlands reported that 47% resulted from E. coli and 24%from group B streptococci. Notice that almost 90% of all cases of E. coli meningitis are caused by the K1 strain,which possesses a capsule identical to that occurring on group B meningococci. Table 1: Escherichia coli Virulence Factors Diarrhea-producing Virulence Factors E. coli Enteroroxigenic E. coli Heat-labile toxin (LT) Heat-stable toxin (ST) Colonization factors (fimbriae) Enterohernorrhagic E. Shiga like toxin (SLT-I) coli Shiga like toxin II (SLF-II) Colonisation factors (fimbriae) Enteroinvasive E. coli Ability to invade epithelial cells Shiga like toxin (SLT-I)? Shiga like toxin II (SLF-II)? Enteropathogenic E. Adhesin factor for epithelial cells coli Urinary trace P- fimbriae infections Meningitis K-1 capsule4. Student’s independent study program 1. General characteristics of Enterobacteriaceae. 2. Structure and tinctorial properties of Escherichia coli. Cultivation. 3. Biochemical properties of E. colі. 4. Peculiarities of antigenic structure of E. colі. Pathogenic serovars of E. coli. 5. Virulence factors of E. colі and their action on organism (pili, colicines, endotoxins, protein toxins). 6. Enteropathogenic, enteroinvasive, enterotoxigenic, enterohemorrhagic E. coli. Diseases they are caused. 6
  7. 7. 7. Methods of diagnosis of colienterites and other diseases, which caused by E. coli. Peculiarities of gettingof tested material for bacteriological examination and its main stages. Significance of serological identification ofcausative agents. 8. Prophylaxis of colienterites.5. Students’ practical activities: 1. To prepare a smear from E. coli, to stain it by Gram’s method and to examine under microscope. 2. To examine the growth perculiarities of E. coli on MPA, Endo’s, Levin’s, Ploskirev’s media, in MPB. 3. To examine biochemical activity of E. coli on Hiss’ media. 4. To carry out presumptive agglutination test with typical sera and culture of an unknown serovar of E. coli. 5. To familiarize with specific diagnostic agglutination Sera. 6. To end diagnostic of inflammatory process causing by staphilococci. Evaluate sensitivity of isolated strainto antibiotics. 6. Control questions and tests Select the correct answers. 1. E. coli has such properties: A – peritrichate; B – Gram-negative; C –does not have flagella; D – gram-positive; E – forms spores. 2. A – grows on Endo’s and Levin’s media; b – on media with lactose the colonies are colour; c – on media with lactose the colonies are colourless; e – does not grow without presence of native protein. 3. A – ferments all carbohydrates of Hiss’ media with production of an acid and gas; b – does not ferment a saccharose; c – produces indole; d – produces hydrogen sulfide; e – fermentats all carbohydrates of Hiss’media with the exception of a saccharose. 3. What antigen do determine serological groups of Escherichia; in what immunological reaction it can be determined? A – K, precipitation test; b – Н, agglutination test; c – O, agglutination test; d – K and Н, agglutination test; e – O, flocculation test. 5. Characteristic of pathogenic serovars: a – enteropathogenic E. coli produce enterotoxins with adenylate cyclase activity; b – enteroinvasive Escherichia coli cause dysentery-like diseases; c – enterotoxigenic Escherichia coli are not capable to parasitize in the cells; d – enterotoxigenic Escherichia coli cause dysentery-like diseases; e – enterotoxigenic Escherichia coli secrete a toxin with adenylate cyclase activity. 6. One can differentiate commensal E. coli from enteropathogenic E. coli using: a – biochemical properties; b –endotoxin production; c – antigenic structure; d –pathogenicity for animals; e – resistance to antibiotics7. List of literature:1.S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, Lugansk, 2004,part 2,chapter24, p. 171-182.I 7