Rods Gram Negative Rods Enteric Rods Enterobacteriacease,Vibrio, Campylobacter,Helicobacter Enterobacteriacease General Characters of Enterobacteriacease (1) Numerous interrelated bacteria flora of intestine (GIT), some of them as normal flora in the vagina (Klebsiella, Proteus) (2) G - rods, non spore forming. (3) Motile with peritrichate flagella, or non motile. (4) Non acid fast. (5) Ferment glucose with or without formation of gas. (6) Nitrates +. (7) Catalase +. (8) Oxidase -. (9) Facultative anaerobes, aerobes. Classification of Enterobacteriacease A- Based on action on lactose:- It s an old method, practical value in diagnostic bacteriology. 1- Lactose fermenter e.g. Escherichia coli, Klebsiella,Citrobacter,Enterobacter 2- Late Lactose fermenter e.g. Shigella sonnei, Serratia 3- Non Lactose fermentation e.g. Salmonella, Shigella, Proteus,Hafnia B- Modern taxonomical concept:-classified into tribes, genera and species by their cultural and biochemical characters. The species are further classified into types, biotypes, serotypes, bacteriophage type and colicin types. At present there are 5 tribes : Tribe I: Escherichia Tribe II: Tribe III: Proteuae Klebsielleae Genus Klebsiella Genus Escherichia Enterobacter Proteus Edwardsiella Hafina Citrobacter Serratia Salmonella Shigella Tribe VI: Tribe V: Yersinae Erwinieae Genus Yersinia Erwinia 1
A- Escherichia coli It lives only in human or animal intestine. Detection of this genus in drinking water is taken as evidence of recent pollution with human or animal excretation. Morphology Gram stain: G - rod,noncapsulated, motile, short (Figure 1). 1 Culture Characters: It s aerobic and facultative anaerobic.in liquid broth shows uniform turbidity after 8-24 hs incubation. On nutrient agar colonies are circular 1-3 mm in diameter smooth, colourless, having entire edge with butyrous consistency and emulsified easily after 12 hs incubation. On MacConkey agar colonies are pink due to lactose fermentation (selective media for all Enterobacteriaceae is MacConkey agar and also its differential media). On blood agar, some strains may show -hemolysis. Biochemical Reactions: It s ferments Lactose, glucose, sucrose, maltose and mannitol forming acid and gas. IMVC reactions show + + - -, urease - and H 2 S -. It can survive for months in soil and water, it killed at 60 C in 20 minutes and chloride(0.5-1 part/million),it s sensitive to streptomycin, tetracycline, chloramphenicol, furadantin, and nalidixic acid. 2
Antigenic Structure There are 4 types of antigens: 1- Somatic antigen (O antigen) = they are heat stable, divided into 164 groups designated as 1, 2, 3 ---- 2- Surface antigen (K antigen) = they are heat-labile, interfere with O agglutination unless destroyed by heating at 100-121 C. They are 3 types: a. L antigen is destroyed by heating at 100 C for 1hs, and it s capacity to combine with antibodies is lost. b. A antigen is capsular antigen and is heat-stable and is associated with wellmarked capsule. c. B antigen is destroyed by heating at 100 C for 1hr, and it s capacity to combine with antibodies is present. 3- Flagellar antigen (H antigen) = they are heat labile, about 75 types have been described. 4- Fimbrial antigen (F antigen) = they are heat labile and have no significance in antigenic classification E. coli. The antigenic pattern of a strain is recorded as number of particular Ag it carries, e.g. O 111 k 58 H 2. The normal colon strain belongs to early group (1, 2, 8, 4 etc) while enteropathogenic belongs to later O group (26, 55, 86, 111 etc). Production of Toxin E. coli produces endotoxin, and produces 2 types of exotoxins which are: a) Enterotoxin which is heat labile, filterable.the mode of action is by raising level of camp in the cells, causing excretion of fluid and electrolytes in the lumen of intestine. The 2 types of E. coli enterotoxin have been: 1- Heat labile toxin LT: is of large molecular weight (80, 000) protein, which gets inactivated by heating at 60 C for 10 mins and their action through mediaton of camp. 2- Heat stable toxin ST: is of low molecular weight (8000 8500) protein, which is not destroyed by heating,non antigenic toxin, seems to simulate fluid secretion in the gut through mediation of cgmp (cyclic guanosine monophosphate). A strain of E. coli may produce one or both types of enterotoxin, which is under genetic control of transmissible plasmids. b) Hemolysin which may be 1- Heat labile, filterable and lethal for animals. 2- Associated with bacterial cell and is not filterable. 3
Pathogenesis A- Gastroenteritis : certain serotypes produce fatal type of gastroenteritis in infants e.g. 4, 26, 46, 55, 86, 111, 112, 119, 127 and 129. Summer diarrhea occurs in children during second and third summer of life in non epidemic form. Lactic acid produced from lactose fermentation may cause irritation of the colon result in violent nausea, vomiting and diarrhea. There are 5 groups of E. coli, which causes diarrhea disease (Figure 2). 2 1- Enteropathogenic E. coli also called EPEC:- They are carrying B type of surface (K Ag), caused several serious intestinal outbreaks of diarrhea in babies less than 18 months old.the pathogenic mecha nism of EPEC is there tight adherence to enterocytes resulting in the loss of microvilli and cupping of enterocytes membrane to bacteria. 2- Enterotoxigenic E. coli also called ETEC:- They produce heat labile and heat stable enterotoxin and thus producing diarrhea in children and traveler s diarrhea. No biochemical markers are available to identity ETEC. They posses surface properties called colonization factors which promote their virulence, and this factors may be pilli or special types of protein K Ag. Most strains of ETEC belong to O serotype 6, 8, 15, 25 etc, they known to cause: Mild or moderately sever childhood diarrhea in developing countries. Cholera life syndrome in adults living in areas where cholera is epidemic. Traveler s diarrhea in persons from developed country that visits developing countries. Outbreaks of diarrhea in newborn nurseries in developed countries. Outbreaks of diarrhea due to fecal contamination of food and water in developing countries. 4
3- Enteroinvasive E. coli also called EIEC:- They invade intestinal epithelium like other dysentery causing bacilli,and it may be late Lac+ or Lac- and may be anaerobic,cause keratoconjunctivitis when instilled in the eyes of guinea pig. They invasion of Hela cells in tissue culture. 4- Enterohemorrhagic E. coli also called EHEC:- there is no fever but haemorrhage is marked. EHEC produces a cytotoxin, it s called verotoxin because it s effects on Vero cells in tissue culture. B- Urinary tract infection (UTI) e.g. cystitis, pyelitis and pyelonephritis. Infection may be precipitated by urinary obstruction due to prostatic enlargement calculi and pregnancy.strains carrying K Ag are responsible for pyelonephritis while strains form cystitis lack K Ag. UTI causing by E. coli binding to epithelial cells receptors by means of adhesion, such as uroepithial adhesion assay has become one of the important parameters. C- Pyogenic infection: e.g. wound infection, abscess, peritonitis, cholecystitis and meningitis. It may cause septicemia. D- Neonatal Meningitis: is a major cause of this disease occurring within the first month of life. The K 1 Ag(capsular type, A type) is particularly associated with such infections. E. Nosocomial (hospital acquired) infections: include sepsis, bacteremia, endotoxic schock and pneumonia. (The most common causes of neonatal meningitis Group B Strep. + E. coli + Listeria. The most common causes of neonatal sepsis with or without meningitis Group B Strep. + E. coli). F- Septicemia: It s one of the commonest causes of septicemia, causes fever, hypotension, disseminated intravascular coagulation (endotoxin). Mortality is significantly high. Laboratory Diagnosis Haematological investigation: Total leukocytes count within normal limits,but in tissue invasion= moderate leukocytosis. Differential leukocyte count polymorphonuclear cells in tissue invasion. Bacteriological investigation: Specimen in UTI mid steam urine is collected under aseptic condition,examined for pus cell, RBC and bacteria. The count of the organism should be more than 100000/ml in UTI, it s called significant bacteriuria. In acute diarrhea a sample of faces or a rectal swab is collected, pus may be collected on sterile condition. 5
Gram strain : shows moderate to large numbers of pus cells and G - bacilli, motile. Cultur: on MacConkey agar shows pink colonies. Biochemical tests: lactose fermenting, IMVC are + + - -. EIEC strains often Lac- and may be detected on MacConkey media,while EHEC, unlike other strains of E coli, ferment sorbitol slowly if at all, and may be detected on MacConkey sorbitol agar. Serological tests ELISA, Precipitin test, radioimmunoassay are other useful tests to establish the identity of enteropathogenic strains. Treatment of faecal matter with fluorescent labeled O group antisera is useful for early diagnosis of diarrhea. DNA probes for different enteropathogenic forms are quite reliable and useful. Treatment and Prevention Prevention by care in selection, preparation and consumption of food and water. Treatment by (1)maintenance of fluid and electrolyte balance is of primary importance in treatment. (2) Extraintestinal disease require antibiotic treatment (Figure 1)and antibiotics sensitivity testing of isolates is necessary to determine the appropriate choice of drugs. It s sensitive to sulfonamides, trimethoprim, tetracyclines, chloramphenicol and aminoglycosides.(3) Nitrofurantoin + nalidixic acid may be useful for treating UTI. (4) In septicemia or serious infection is required to be started treated immediately without waiting for drug sensitivity tests as gentamicin. (5)Preparation of vaccine against E coli colonization factor Ag (CFA) is being considered( CFAII, IV and I). B- Klebsiella Short, thick, large,g- rod,non motile, capsulated. It s growing on ordinary media forming large mucoid colonies of varying degree of stickiness. They are Lac + on MacConkey agar. Ferment carbohydrate forming acid and gas. IMVC are - - + +. Urease +. It s found in the mucosa of upper respiratory tract, intestine, and genitourinary tract. Most spps are pathogenic and classification depends on biochemical reactions which vary depends on capsular type, like spps below: Klebsiella pneumonia It ferment sugar glucose, lactose, mannitol with production of acid and gas. IMVC are - - + +, Urease +. It causes labor pneumonia, sinusitis, otitis media, meningitis.it also causes urinary tract infections and bacteremia, particularly in hospitalized patients. K. pneumoniae and K. oxytoca cause necrotizing labor pneumonia in individuals compromised by alcoholism, diabetes, or chronic obstructive pulmonary disease. 6
Klebsiella ozaenae It causes foul smelling nasal discharge (ozaena). Biochemical reaction are variable, it capsulated.other strains cause granulomatous lesion of nose in Mediterranean countries. Klebsiella rhinosclermatis Cause rhinoscleroma disease. Organism are seen intracellular in lesion, it capsulated. K. erogenes Usually present in human intestine and isolated from faeces in small number than E coli, so many survive longer out of intestine. K. cloasa It differs in motility +, not always capsulated, liquefied gelatin. C- Proteus It s G - rods showing great variation in size, it may be in long filaments or in granular form, actively motile, non capsulated. It s aerobic and facultative anaerobic. In broth media shows uniform and moderate turbidity, powdery deposit and ammonical odor after 18-24 hs. On MacConkey agar shows Lac - with fishing or bad odor. On nutrient agar shows swarming motility best seen at 20 C, while P. vulgaris and P. mirabilis swarm on solid media at 37 C after 12-18 hs incubation. Swarming may be due to progressive surface growth spreading from the edge of parent colony. This phenomena does not occur on MacConkey agar. The biochemical reactions of K. spps are: glucose Lactose Urease H 2 S I M V C P. vulgaris + - + + + + - +d P. mirabilis + - + + - + - P. reltegri ± - + - + + - + P. morgani +d - + - + + - - It forms acid and gas from glucose (except P. reltegri) Deaminates phenylalanine to phenyl pyruvic acid. Hydrolysis of urea by urease enzyme in few minutes. All of the them nitrate +, Lactose, MV are + -, H 2 S + only in P. vulgaris and P. mirabilis, Indole + (except P. mirabilis), Citrate + (except P. morgani),see the table above. 7
Antigenic Structure A number of O and H antigen are produced in P. vulgaris. Pathogenecity UTI, pyogenic lesions like abscess, wound infection, respiratory tract infections, diarrhea by P. morgani. P. spps common causes of uncomplicated as well as nosocomial UTIs. Other extraintestinal infections such as wound infection, pneumonias, and septicemias are associated with comporomised patients. Laboratory diagnosis Hematological investigation leukocytosis with increase in PMN cells. Bacteriological investigation (see page7) On culture of material (urine, pus, sputum etc).we find swarming type of growth.it can be further identified by biochemical tests. D- Shigella Causes shigellosis (basillary dysentery) a human intestinal disease that occurs commonly among young children. Shigella is G- rod, non motile,noncapsulated, and Lac -, only S. sonnei is late Lac+. Epidemiology It typically spread from person to person with contaminated stools, serving as a major source of organism. Flies and contamined food or water can also transmit the disease. Shigellosis has a low infectious dose (fewer than 200 viable organisms). Hence, secondary cases within a house-hold are common under crowding conditions or poor sanitation. The forty serotypes of Shigella are organized into 4 groups (A, B, C, D) based on their polysaccharide O-Ag. Group D (S. sonnei) is the serogroup found most commonly in U.S. Pathogenesis and Clinical Significance It s invade and destroy the mucosa of the large intestine. Infection rarely penetrates to deeper layers of the intestine, and does not lead to shigella bacteremia (Figure 1). 8
1 An exotoxin with enterotoxic and cytotoxic properties has been isolated from these organisms, and it s toxicity may play a secondary role in development of intestinal lesions. It s cause classic dysentery, characterized by bloody diarrhea, mucus and painful abdominal cramping. The disease is most severe in the very young and elderly individuals, in whom shigellosis may lead to severe dehydration and some time death. Laboratory Identification Gram stain G- rods, non motile, non capsulated (Figure 2).Cultured from stools using differential selective media, like Hektoen agar, or media specific for intestinal pathogens such as MacConkey s agar or Deoxycholate citrate agar ( DCA) on which they form colorless colonies due to absence of lactose ferments, only S. sonnei give pale pink colonies which ferment lactose late( Late Lac+) on both media. On Salmonella Shigella Agar,colorless colonies without black center. Biochemical reactions glucose + acid only, nitrate +, Lac - (except S. sonnei), I M VC are + + - - (like E coli), H 2 S -,uerase-, Catalase +, mannitol+ Treatment and Prevention Antibiotics (ciprofloxacin, azithromycin) can reduce the duration of illness. Protection of the water and food supply and personal hygiene are important (Figure 2). 9
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