General characters 1-Members of this family are found mostly in the intestinal tract of man and animals as normal flora or as pathogens 2-They are Gram –ve, relatively small size and non-spore forming rods 3-Capsule production (Klebsiella pneumoniae, and enterobacter aerogenes) give rise a large mucoid colonies 4-Members of this family are primarily motile with peritrichous flagella although some strains are non-motile 5-Several strains possess fimbriae or pili 6-Grows readily onto ordinary media under aerobic and facultative anaerobic

7-The genera within the family on the basis of biochemical characteristics as: Capacity to ferment specific carbohydrates Utilize of citrate Indole and urea tests Hydrogen sulfide test 8-The genus distinguished only by serological procedures into numerous serotypes (serovars) using Kauffman-White Scheme 9-All species ferment glucose, nitrate are reduce to nitrite and oxidase is not produced.

On MacConkey`s media *Lactose fermenter (Coliform) as E.coli, Citrobacter, Klebsiella *Intermediate (Late fermentation) (Paracolon): As Arizona, Serratia, Hafnia *Lactose not ferment: As Proteus, Salmonella, Providence, Shigella, Morganella

G.: Escherichia (E.coli( G.: Escherichia (E.coli( Gr-ve bacilli, arranged in single, pairs or groups Size: 2-3x µ Motile by peritrichous flagella Capsulated by polysaccharide capsule Non sporulated Morphology

Gram –ve bacilliPeritrichous flagella of E.coli

Culture characters  Aerobic and facultative anaerobic  Grow on ordinary media, optimum temperature at 37°C (for man and animals), it can grow at wide range of temperature (20-44)  Colonies are circular outline, smooth, glistening, transparent, unpigmented.  MaCconkey agar: Pink colonies (lactose fermenter) Selective media for E.coli: Endomedia, Jaster media Eosin Methylene Blue EMB Leiven media MaCconkey agar Kauffman media Brillient green media

E.Coli on MaCconkey agar Pink colonies Lactose fermenter E.Coli on EMB media Yellow green colonies

Biochemical reactions Sugar fermentation: glucose and lactose (acid and gas) Citrate –ve H2S: -ve Urea hydrolysis: -ve Gelatin liquefaction: +ve Litmus milk test: +ve (acid and clot) Indole: +ve V.P.: -ve M.R.: +ve I M V C

Indole +ve: Pink ring Indole –ve: Yellow ring Citrate +ve: blue colour Citrate –ve: green colour

M.R. +ve: Red colour M.R. –ve: Yellow colour Voges-proskauer +ve: Pink ring Voges-proskauer –ve: No ring

E.Coli on Triple Sugar Iron(TSI): Y⁄ Y Gas production Urease test +ve: purple colour Urease test –ve: yellow colour

Antigenic structure 171 types It composed of polysaccharide phospholipid- protein complex Heat stable (not destroyed at °C) Not destroyed by alcohol 80 types It composed of polysaccharide L, B Ags: inactivated by heating at 100°C for an hour A Ag : inactivated by heating at 121°C for 2.5 hours Somatic Ag Envelop or capsular Ag O Ag K Ag

H Ag 56 types It composed of protein called flagellin F Ag K88: F4 K99: F5 It composed of glycoprotein Heat labile Acts as adhesion or colonization factors, indicate virulence Flagellar Ag Fimbrial Ag

Toxins Exotoxin Enterotoxin that composed of heat labile and heat stable fractions Endotoxin Vomition, diarrhea, fever Colicin Antibiotic like substance that produced from virulent strains of E.coli It has bactericidal effect causing death for M.O. but not used for treatment as toxic for tissues

Resistanc e M.O.can survive for weeks or months in water, faecal matter especially if away from sun light Destroyed at 55°C for an hour, 60°C for 20 minutes (vegetative M.O.) Relatively susceptible to physical and chemical agents Susceptible to lethal action of phenol, cresol and lysol

Man Food poisoning Factors affecting pathogenicity Polysaccharide somatic Ag Fimbrial Ag Capsular Ag Enterotoxins, shiga like toxin Sidrophore (combete with iron of host cell) Pathogenicity

Clinical symptom Microscopical examination Culture characters Biochemical reaction Serological tests Diagnosi s Slide agglutination Tube agglutination

Coli titer Minimum amount of fluid or Gram of solid sample that contain one E.coli cell Coli index Amount of E.coli bacterial cells that present in one liter (fluid) or one Kg (solid) sample

Klebsiella Morphology Gr-ve bacilli, arranged in single, pairs or short chains Size: 1-3x0.5 µ Non motile Capsulated by polysaccharide capsule give rise to mucoid colonies Non sporulated

 Aerobic and facultative anaerobic, optimum temperature 37°C  Colonies are circular outline, smooth, glistening, transparent, unpigmented, 1-4 mm in diameter, mucoid due to present of capsule but after repeated subculture, it lost mucoid capsule and colonies become smaller in size  On MaCconkey agar: Pink colonies (lactose fermenter)  On blood agar: No haemolysis Culture characters

Klebsiella mucoid colonies Klebsiella on EMB Large mucoid pink to purple colonies with no metallic green sheen

Biochemical reactions  Sugar fermentation: glucose, lactose, sucrose and salicin (acid and gas)  Citrate: +ve  Urea hydrolysis: +ve (slowly after 4 days)  H2S: -ve  Indole: -ve  Gelatin liquefaction: -ve  Litmus milk test: -ve  V.P.: +ve  M.R.: -ve I M V C

Indole –ve Citrate +ve V.P. +ve M.R. –ve Urease +ve

Species K.pneumoniae K.arerogenous: K.rhinoscleromatis K.ozaenae Causing: Some members are present normally as normal flora in intestine and respiratory tract. K.Pneumoniae: sever Pneumonea, Urinary tract infection K.rhinoscleromatis: rhinoscleroma, chronic granulomatous condition affecting mucous membrane of nose, throat extended to larynx K.Ozaenae : atrophic rhinitis, nasal bad odour K. aerogenous: Urinary tract infection

Shigella Morphology Gr -ve small rods Size: 2-3 x 0.5 µ Non motile Non sporulated Non capsulated Culture characters 0n MaCconkey media:pale colonies (non lactose fermenter)

Biochemical reactions Sugar fermentation: Sucrose non lactose fermenter: subgroups A, B, C Lactose non sucrose fermenter: subgroup D Citrate: -ve Urea: -ve H2S: -ve Indole: -ve M.R.: +ve V.P.: -ve Gelatin liquefaction: -ve I M V C

Species Sh.dysentery (A) Sh. Sonnei (D) Sh.flexneri (B) Sh.boydii (C) Causing: Human bacillary dysentery especially in children

Salmonella Morphology Gram-ve short rods, arranged singly, in pairs or groups Size: 2-4 x 0.5µ Motile by peritrichous flagella except S.pullorum and S.gallinarum Sometimes capsulated (polysaccharide capsule) Non sporulated

Culture characters * Aerobic and facultative anaerobic * Colonies are round, smooth and transperant 2-3mm For isolation it require Enrichment media as: Selenite-F- broth Tetrathionate broth Incubation at 37°C hours Solid media Followed by Brillient green agar Desoxycholate citrate agar Xylose lysin desoxycholate (Salmonella Shigella agar MaCconkey agar Incubation at 37°C hours

Non lactose fermenter On MaCconkey agar Pale colonies Salmonella on XLD media Red colonies with black center

Salmonella 0n Brillient green agar Yellow to green colonies Salmonella on SS agar

Biochemical reactions Sugar fermentation: It ferment sugars with acid and gas except lactose, sucrose and salicin Citrate +ve H2S: +ve Urea hydrolysis: -ve Gelatin liquefaction: -ve Nitrate: +ve Indole: -ve V.P.: -ve M.R.: +ve I M V C - +

Citrate +ve Indole –ve Urease-ve M.R. +ve V.P. -ve

TSI media **Alkaline (Red colour) **Acid (Yellow colour) Glucose fermenter **Black colour (H2S production) **Gases (due to fermentation)

Antigenic structure O Ag: Thermostable Designated by arabic numbers H Ag: Phase I: More specific Designated by small letters of alphabetic Phase II: Less specific Designated by arabic numbers K or envelope Ag Vi Ag M or mucoid Ag

Kauffman-White scheme: Classify Salmonella into different O- groups each of which contain No. of serotypes have common O Ag not found in the other O- group. It designated by capital letters from A to Z Groups A to E nearly contain all Salmonella species that are important pathogens for man and animal Identification of serotypes by tube agglutination test It was found that: Addition of alcohol destroy H Ag to prepare O Ag Addition of formalin destroy O Ag to prepare H Ag Boiling for minutes destroy Vi Ag

H Ag Phase I Phase II O AgGroupSerotype -a1,2,12AS.paratyphi A 1,2b1,4,5,12BS.paratyphi B 1,2i1,4,5,12BS.typhimurium 1,5c6,7CS.cholera-suis 1,2e, h6,8CS.newport 1,6c4,12BS.abortus ovis enx-4,12BS. abortus equi -c,m1,9,12DS.enteritidis --1,9,12DS.Pullorum and S.gallinarum Kauffman-White scheme:

Pathogenicity Classification: A. Salmonella causing enteric fever 1. S.typhi 2. S.paratyphi A 3. S.paratyphi B 4. S.paratyphi C B. Salmonealla causing food poisoning 1. S.Typhi murium 2. S.Enteritidis C. Salmonella causing septicemia 1. S.cholerae-suis 2. S.virchow

Enteric fever Pathogenicity: Source of infection: Human case and Human carrier (Urine and stool) Mode of transmission: contamination food and water.

1. M.O enter body via mouth multiplies in payer’s patches of small intestine via lymphatic into mesenteric Ln, spleen, liver this called incubation period which still (14 days) 2. M.O pass to blood and produced bacteremia with first appearance of symptoms and this called (First week) 3. M.O stimulate immune system to produced Ab and this called (2nd week).

3. M.O disappear from blood to internal organ, liver, gall bladder, spleen, kidney to be extracted in intestine again and M.O excreted in stool and urine this called (3rd week). 4. M.O are localized in peyer’s patches with typical pathology which include Necrosis, ulceration and proliferation of intestine in sever case.

Laboratory Human case: A. First week 1. sample: (stool) 2. Direct film stain by Gram stain: G-ve bacilli, motile. Non-spore, non-capsulated 3. culture characters: Blood culture 5-10ml of venous blood are added to 50 ml of broth containing 0.5% Na+ taurocholate and incubation at 37C/24h. And the subculture on:

a. MacConky’s medium pale yellow colonies b. DCA (desoxy cholate citrate agar) pale yellow c. S-S pale yellow d. XLD (xyline lysine dextrose agar) pink colonies Cloteculture: 5ml of venous blood are left to clot and then clote is added to 15 ml of bile salt broth the incubation 37C/24h. And subculture

2 nd week: By Widal test 3 rd week: sample (stool or urine) Stool sample riched by different type of M.O so to detected salmonella must be 5-10 ml of stool is added on (selneite broth) where kill all organismis except salmonella and shigella. Incubation 37C/24h and subculture

Human carrier Sample: stool or urine Direct film Culture character: At first: M.O present in gall bladder (chola- mate) Patient give laxative diarrhea M.O in large amount in stool. Media: 5-10ml of stool+ salenite broth 37C/24h, subclture. Urine centerfugation then deposite is taken and examined

Clinical symptom Microscopical examination Culture characters Biochemical reaction Serological tests Diagnosi s Slide agglutination Tube agglutination Rapid agglutination Whole blood agglutination