Gram Negative Rods Part 1

E.coli Enterobacter Proteus spp. Klebsiella spp. Serratia

CONTENTS
Species and pathogenicity
E.coli Enterobacter Proteus spp. Klebsiella spp. Serratia Microscopy Culture Biochemical reaction Antimicrobial sensitivity

Laboratory diagnosis

Species and Pathogenicity

Enterobactericae
Large family of Gram negative rod Facultative anaerobic bacilli On gram stain-coccobacilli or as straight rod Found in colon as part of normal flora Cause variety of diseases with different pathogenic mechanisms on nonselective media eg sheep blood agar(SBA) or chocolate(CHOC) value little identification Differential and selective media-mac conkey and hektoen entric(HE) and xylose lysin deoxycholate (XLD), contain lactose and sucrose (produce H2S- blackening) Fermentation color changes on the medium(pink) on Mac conkey

 Gram negative rod

Mobile with peritrichate flagella.

Some are non-motile (Klebsiella, Shigella).

Medical significant of

E. coli, Klebsiella, Enterobacter, Serratia, Proteus, Salmonella, Shigella, Campylobacter, Helicobacter and Vibrio

Virulence and antigenic factors

Virulence- ability to adhere, colonize, produce toxins and invade tissue AntigenO antigen/somatic antigen-heat stable antigen-located on cell wall H antigen/flagellar antigen-heat labile antigen found on the surface of flagellar- motility K antigen/capsular antigen-heat labile polysaccharide found on encapsulated. E coli, Salmonella

Family Enterobacteriaceae

Ingestion contaminated food

Genus Escherichia Gram negative bacilli Isolated colon flora Fecal contamination Selective (Mac Conkey) differential media (EMB) Lactose positive(pink)-surrounding area of precipitate bile salt on mac conkey, EMB-green metallic sheen Posses adhesive fimbriae and sex pili and O,Hand K antigen Large intestine, recongnized as UTIs-human and animal

E.coli

Escherichia Coli
Normal flora Opportunistic

pathogen

Pathogen
5 major diarrheagenic

Characteristic
Fermentation of glucose, lactose, trehalose and xylose Indole production from tryptophane Methyl red positive Voges-proskauer negative Do not produce H2s, Dnase, urease, phenylalamine deaminase Nitrate to nitrite

 Common causes of septicemia and meningitis in neonates 40% of gram negative meningitis cases

Newborn acquires infection in birth canal just before or during delivery When mothers vagina is heavily colonized

Klebsiella
Gram-negative bacilli, short, non-motile, capsulate. Commensal in intestine Klebsielleae Klebsiella pneumoniae causes: Fried-Lander pneumonia (severe fatal bronchopneumonia). Neonatal meningitis. Lung abscess. Wound infection UTI

Characteristic
Most grow on simmons citrate and potassium cyanide broth Not produce hydrogen sulfide A few hydrolyze urea slowly Methyl red positive Voges proskauer test negative Indole not produce Motility is variable Pink-mac conkey Large and very mucoid colonies, , moist and gray colonies on non selective media

 Motile Colony morphology resembles Klebsiella on MAC Most commonly isolated species E. cloacae E. aerogenes Both have been isolated from wounds, urine, blood and CSF

 Opportunistic pathogens associated with nosocomial outbreaks Ferments lactose slowly Resistant to multiple antibiotics S. marcescens Species that is clinically important Produces characteristic pink to red pigment (prodigiosin) Cause hospital acquired infections of urinary or respiratory tract Bacteremic outbreaks in nurseries and cardiac surgery and burn units

Normal intestinal flora Opportunistic pathogens Cannot ferment lactose 4 species

2 species human pathogens
P. mirabilis P. vulgaris Have been isolated from urine, wounds and ear and bacteremic infections

 Cause nosocomial UTIs

Ascend urinary tract causing infection in both lower and upper urinary tract Can infect proximal kidney tubules cause AGN, particularly in patients with urinary tract defects or catherization

Laboratory Diagnosis

LAB diagnosis

Laboratory Diagnosis of E. coli Diarrhea

Specimen: Stool, examined as follows: 1. Microscopic examination of fresh preparation 2. Isolation on MacConkeys agar plates, 3. Identification by sugar fermentation.

Laboratory Diagnosis of Klebsiella infections

Specimens: Sputum, urine, pus. Microscopy: Grams stain shows short Gram negative rods with unstained halo around them due to the presence of the capsule. Isolation: On blood agar and MacConkeys agar media, it shows mucoid lactose fermenting colonies.

Genus : Proteus
Laboratory diagnosis of Proteus infections:
Specimen: urine, pus, etc Microscopy: Gram-negative rods. Culture: on blood agar medium swarming growth. on MacConkeys agar non-lactose fermenting colonies.

Identification: sugar fermentation rapid urease activity.

Swarming Growth

E. Coli (lac +)

Klebsiella pneumoniae (lac +)

Enterobacter aerogenes (lac +)

Serratia marcescens (lac -)

Proteus vulgaris (lac neg)

Prodigiosin

 Differential agar slants - contain more than one sugar, and possess other indicators.
In these slants both respiration (aerobic) and fermentation (anerobic) growth can take place. Very useful media for distinguishing among this group of organisms.

Double Sugar Iron Agar slant

this media contains lactose and glucose, in a ratio of 10 lactose molecules to 1 glucose molecule, a ferric salt, and phenol red as indicator (yellow below pH7, red above).

It is inoculated by stabbing into the butt of the tube with a needle, withdrawing the needle and streaking the surface of the slant

Result
SLANT COLOR:Interpretation RED: does not ferment either lactose or sucrose YELLOW: ferments lactose and/or sucrose BUTT COLOR/CONDITION Interpretation RED: no fermentation of glucose YELLOW: some fermentation of glucose has occurred, acid has been produced GAS FORMED : Seen as cracks in the agar, bubbles, or the entire slant may be pushed out of the tube. BLACK: H2S has been produced

 Facultative anaerobe Ferments glucose and lactose as a carbon source Produces Gas Does not produce hydrogen sulfide

 Facultative anaerobe Ferments glucose & lactose as a carbon source Produces gas Does not produce hydrogen sulfide

 Facultative anaerobe Ferments glucose & lactose as a carbon source Produces gas Does not produce hydrogen sulfide

 Can ferment glucose but not lactose as carbon source Produces hydrogen sulfide

 Yet, another set of media to differentiate among these genera is the commercially available Enterotube II (Roche Diagnostics, Montclair, N.J), which contains biochemical tests in miniature form.

Note: the percentage shown above a test in the images below indicates the likelihood (as percent) that a particular colony /clone of that specific organism (genus, species) would yield a positive test.

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