the enterobacteriaceae basic properties.ppsx x

THE ENTEROBACTERIACE

AEBASIC PROPERTIES

Nursing Pathwww.drjayeshpatidar.blogspot.com

Characteristics of the Enterobacteriaceae

Gram-negative rods Glucose is fermented with strong

acid formation and often gas Cytochrome oxidase activity is

negative Nitrate is reduced to nitrite

www.drjayeshpatidar.blogspot.in

Gram’s Stain for Bacterial Morphology

Crystal violet binds to cell wall peptidoglycan with Gram’s iodine as a mordant

Safranin or basic fuchsin counterstains bacterial cells decolorized by alcohol-acetone


Gram’s Stain for Bacterial Morphology

Thick cell-wall peptidoglycan layer of gram-positive bacteria strongly binds crystal violet and resists decolorization by alcohol-acetone

Thin cell-wall peptidoglycan layer of gram-negative bacteria located beneath a thick lipid-rich outer membrane weakly binds crystal violet that is readily removed by alcohol-acetone decolorization


Gram’s Stain Procedure Flood surface of smear with crystal violet

solution After 1 min thoroughly rinse with cold tap

water Flood smear with Gram’s iodine for 1 min Rinse smear with acetone-alcohol

decolorizer until no more crystal violet in rinse effluent

Rinse with cold tap water Flood smear with safranin (regular Gram’s

stain) or basic fuchsin (enhanced Gram’s stain)

Rinse with cold tap water Dry smear in slide rack Microscopically examine stained smear

using oil-immersion light microscopywww.drjayeshpatidar.blogspot.in

Glucose Fermentation Oxidation-reduction of glucose in the absence of

molecular oxygen (anaerobic glycolysis) Energy from hydrolysis of chemical bonds in anaerobic

glycolysis captured as high energy phosphate bonds of adenosine triphosphate (ATP)

NAD is reduced to NADH2 by accepting electrons during glycolytic conversion of glucose to pyruvate

NADH2 in turn reduces pyruvate with oxidation of NADH2 to NAD which supports continued anaerobic glycolysis, and generation from pyruvate of alcohols, carboxylic acids, and CO2 gas

End products of glucose fermentation: organic acids and CO2 gas

Fermentation detected by acidification of glucose-containing broth (color change in broth or agar medium containing pH indicators), and (for aerogenic species) production of gas (fractures in agar, gas bubbles in inverted Durham tube)

pH indicators: phenol red (yellow at acid pH), methyl red (red at acid pH), neutral red (red at acid pH), bromcresol purple (yellow at acid pH)www.drjayeshpatidar.blogspot.in

Spot Cytochrome Oxidase Test

The spot cytochrome oxidase test is the first test performed with gram-negative bacteria recovered in culture

The optimal plate medium for a spot cytochrome oxidase test is a trypticase soy agar (TSA) containing 5% sheep blood

Bacterial colonies should be 18 to 24 hr old


Spot Cytochrome Oxidase Test

In a positive test, bacterial cytochrome oxidase oxidizes the colorless reduced substrate tetramethyl-p-phenylenediamine dihydrochloride (TPDD) forming a dark purple oxidized indophenol product

Streak a small portion of bacterial colony to filter paper soaked with a 1% solution of TPDD

If the streak mark turns purple in 10 sec or less, the spot oxidase test is interpreted as positive www.drjayeshpatidar.blogspot.in

Nitrate Reduction Enterobacteriaceae extract oxygen

from nitrate (NO3) producing nitrite (NO2)

NO2 detected by reaction with α-naphthylamine and sulfanilic acid producing a red colored complex

Absence of red color indicates either no reduction of NO3 or reduction to products other than NO2 (denitrification)

Confirmation of true negative test: addition of zinc ions which reduce NO3 to NO2 producing a red color in the presence of α-naphthylamine and sulfanilic acid


Enterobacteriaceae: Genetic Properties

Chromosomal DNA has 39-59% guanine-plus-cytosine (G+C) content

Escherichia coli is the type genus and species of the Enterobacteriaceae

Species of Enterobacteriaceae more closely related by evolutionary distance to Escherichia coli than to organisms of other families (Pseudomonadaceae, Aeromonadaceae)


Enterobacteriaceae: Major Genera

Escherichia Shigella Salmonella Edwardsiella Citrobacter Yersinia Klebsiella Enterobacter Serratia Proteus Morganella Providencia


Enterobacteriaceae: Microbiological Properties

Gram-negative and rod shaped (bacilli)

Ferment rather than oxidize D-glucose with acid and (often) gas production

Reduce nitrate to nitrite Grow readily on 5% sheep blood or

chocolate agar in carbon dioxide or ambient air

Grow anaerobically (facultative anaerobes)www.drjayeshpatidar.blogspot.in

Enterobacteriaceae: Microbiological Properties

Catalase positive and cytochrome oxidase negative

Grow readily on MacConkey (MAC) and eosin methylene blue (EMB) agars

Grow readily at 35oC except Yersinia (25o-30oC)

Motile by peritrichous flagella except Shigella and Klebsiella which are non-motile

Do not form sporeswww.drjayeshpatidar.blogspot.in

Enterobacteriaceae: Natural Habitats

Environmental sites (soil, water, and plants)

Intestines of humans and animals


Enterobacteriaceae: Modes of Infection

Contaminated food and water (Salmonella spp., Shigella spp., Yersinia enterocolitica, Escherichia coli O157:H7)

Endogenous (urinary tract infection, primary bacterial peritonitis, abdominal abscess)

Abnormal host colonization (nosocomial pneumonia)

Transfer between debilitated patients Insect (flea) vector (unique for

Yersinia pestis)www.drjayeshpatidar.blogspot.in

Enterobacteriaceae: Types of Infectious Disease

Intestinal (diarrheal) infection Extraintestinal infection

Urinary tract (primarily cystitis)Respiratory (nosocomial

pneumonia)Wound (surgical wound

infection)Bloodstream (gram-negative bacteremia)Central nervous system

(neonatal meningitis)www.drjayeshpatidar.blogspot.in

Enterobacteriaceae: Urinary Tract Infection, Pneumonia

Urinary tract infection: Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., and Proteus mirabilis

Pneumonia: Enterobacter spp., Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis


Enterobacteriaceae: Wound Infection, Bacteremia

Wound Infection: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis

Bacteremia: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis


Enterobacteriaceae: Nosocomial Infections in the United States 1986-1989 and 1990-19961

Escherichia coli 27,871 (13.7%)

Enterobacter spp. 12,757 (6.2%)

Klebsiella pneumoniae 11,015 (5.4%)

Proteus mirabilis 4,662 (2.3%)

Serratia marcescens 3,010 (1.5%)

Citrobacter spp. 2,912 (1.4%)

1Enteric Reference Laboratory, Centers for

Disease Control and Prevention


Enterobacteriaceae: Intestinal Infection

Shigella sonnei (serogroup D) Salmonella serotype Enteritidis Salmonella serotype Typhimurium Shigella flexneri (serogroup B) Escherichia coli O157:H7 Yersinia enterocolitica


Triple Sugar Iron (TSI) Agar

Yeast extract 0.3% (% = grams/100 mL)

Beef extract 0.3% Peptone 1.5% Proteose peptone 0.5%

Total Protein = 2.6% Lactose 1.0% Sucrose1 1.0% Glucose 0.1%

Carbohydrate = 2.1%1Absent in Kligler Iron Agar


Triple Sugar Iron (TSI) Agar

Ferrous sulfate Sodium thiosulfate Sodium chloride Agar (1.2%) Phenol red pH = 7.4


TSI Reactions of the Enterobacteriaceae

Yellow deep, purple slant: acid deep due to glucose fermentation , no lactose or sucrose fermentation with alkaline slant due to production of amine’s from protein

Black deep, purple slant: acid deep due to glucose fermentation with H2S production, no lactose or sucrose fermentation

Yellow deep and slant: acid deep and slant due to glucose as well as lactose and/or sucrose fermentation

Black deep and yellow or black slant: acid deep and slant with glucose and lactose and/or sucrose fermentation with H2S production

Fracturing or lifting of agar from base of culture tube: CO2 production


TSI Reactions of the Enterobacteriaceae

A/A + g = acid/acid plus gas (CO2) A/A = acid/acid A/A + g, H2S = acid/acid plus gas, H2S Alk/A = alkaline/acid Alk/A + g = alkaline/acid plus gas Alk/A + g, H2S = alkaline/acid plus

gas, H2S Alk/A + g, H2S (w) = alkaline/acid

plus gas, H2S (weak)www.drjayeshpatidar.blogspot.in

A/A + g Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca Enterobacter aerogenes Enterobacter cloacae Serratia marcescens1, 2

1Non-lactose, sucrose fermenter255% + g


A/A Serratia marcescens1, 2

Yersinia enterocolitica2

145% of strains2Non-lactose, sucrose fermenter


A/A + g, H2S

Citrobacter freundii Proteus vulgaris1

1Non-lactose, sucrose fermenter


Alk/A Shigella Providencia


Alk/A + g Salmonella serotype Paratyphi A


Alk/A + g, H2S

Salmonella (most serotypes) Proteus mirabilis Edwardsiella tarda


Alk/A + g, H2S (w)

Salmonella serotype Typhi


MacConkey (MAC) Agar Peptone 1.7% Polypeptone 0.3% Lactose1 1.0% Bile salts2 0.15% Crystal violet2

Neutral red3

Sodium chloride 0.5% Agar 1.35% pH=7.1

1Differential medium for lactose fermentation

2Inhibit gram positives and fastidious gram-negatives; MAC agar selective for

gram-negatives

3Red color at pH < 6.8


Eosin Methylene Blue (EMB) Agar (Levine)

Peptone 1.0% Lactose1 0.5% Eosin y2

Methylene blue2

Agar pH = 7.2

1Modified formula also contains sucrose (0.5%)

2Inhibit gram-positives and fastidious gram-negatives; selective

for gram-negatives. Eosin y and methylene blue form a

precipitate at acid pH; differential for lactose fermentation


Bacterial Utilization of Lactose

Presence of β-galactoside permease: Transport of β-galactoside (lactose) across the bacterial cell wall

Presence of β-galactosidase: Hydrolysis of β-galactoside bond (lactoseglucose + galactose)

ONPG: Orthonitrophenyl-β-D-galacto-pyranosidewww.drjayeshpatidar.blogspot.in

Differential Reactions of the Enterobacteriaceae by TSI, ONPG, and MAC

Escherichia coli Red colonies,(A/A, ONPG+) pitted

Klebsiella1 Red colonies,(A/A, ONPG+) mucoid

Enterobacter Red colonies(A/A, ONPG+)

Citrobacter2 Red or colorless(A/A or Alk/A, ONPG+) colonies

Serratia Colorless colonies(A/A, ONPG+)

1K. pneumoniae, indole –, K. oxytoca, indole +2C. freundii, indole – and H2S +, C. koseri, indole + and

H2S –www.drjayeshpatidar.blogspot.in

Differential Reactions of the Enterobacteriaceae by TSI, ONPG, and MAC

Shigella Colorless Colonies(Alk/A; ONPG – A, B, and C1; ONPG + D1)

Salmonella Colorless Colonies(Alk/A + H2S; ONPG –)

Proteus Colorless Colonies(Alk/A + H2S2; ONPG –)

Edwardsiella tarda Colorless Colonies(Alk/A + H2S; ONPG–)

Yersinia Colorless Colonies(A/A, ONPG +)

1Shigella A, B, and C, ornithine –; Shigella D, ornithine +2Proteus mirabilis. P. vulgaris sucrose + with A/A + H2S on TSI


Differential Reactions of the Enterobacteriaceae by EMB

Escherichia coli Colonies with metallic green sheen

Klebsiella Colonies with precipitate (ppt) and mucoid

appearance Enterobacter Colonies with ppt Citrobacter Colonies with/without ppt Serratia Colonies without ppt Shigella Colonies without ppt Salmonella Colonies without ppt Proteus Colonies without ppt Yersinia Colonies without ppt


ONPG Reaction and Lactose Fermentation (Lac)

ONPG Lac

Escherichia coli + +

Shigella sonnei + –

Citrobacter + +/–

Yersinia enterocolitica + –

Klebsiella + +Serratia marcescens +

–


Xylose Lysine Deoxycholate (XLD) Agar: Composition

Xylose 0.35% Lysine 0.5% Lactose 0.75% Sucrose 0.75% Sodium chloride 0.5% Yeast extract 0.3% Sodium deoxycholate 0.25% Sodium thiosulfate Ferric ammonium citrate Agar 1.35% Phenol red pH = 7.4


XLD Agar: Growth of Salmonella

Salmonella selective due to bile salt. Xylose fermentation (except Salmonella

serotype Paratyphi A) acidifies agar activating lysine decarboxylase. With xylose depletion fermentation ceases, and colonies of Salmonella (except S. Paratyphi A) alkalinize the agar due to amines from lysine decarboxylation.

Xylose fermentation provides H+ for H2S production (except S. Paratyphi A).


XLD Agar: Appearance of Salmonella

Ferric ammonium citrate present in XLD agar reacts with H2S gas and forms black precipitates within colonies of Salmonella.

Agar becomes red-purple due to alkaline pH produced by amines.

Back colonies growing on red-purple agar-presumptive for Salmonella.


XLD Agar: Growth of Escherichia coli and Klebsiella pneumoniae

Escherichia coli and Klebsiella pneumoniae are lysine-positive coliforms that are also lactoseand sucrose fermenters. The high lactose and sucrose concentrations result in strong acid production, which quenches amines produced by lysine decarboxylation. Colonies and agar appear bright yellow. Neither Escherichia coli nor Klebsiella pneumoniae produce H2S.


XLD Agar: Growth of Shigella and Proteus

Shigella species do not ferment xylose, lactose, and sucrose, do not decarboxylate lysine, and do not produce H2S. Colonies appear colorless.

Proteus mirabilis ferments xylose, and thereby provides H+ for H2S production. Colonies appear black on an agar unchanged in color (Proteus deaminates rather than decarboxylates amino acids). Proteus vulgaris ferments sucrose, and colonies appear black on a yellow agar.


Hektoen Enteric (HE) Agar: Composition

Peptone 1.2% Yeast extract 0.3% Bile salts 0.9% Lactose 1.2% Sucrose 1.2% Salicin 0.2% Sodium chloride 0.5% Ferric ammonium citrate Acid fuchsin Thymol blue Agar 1.4% pH = 7.6


HE Agar: Growth of Enteric Pathogens and Commensals

High bile salt concentration inhibits growth of gram-positive and gram-negative intestinal commensals, and thereby selects for pathogenic Salmonella (bile-resistant growth) present in fecal specimens.

Salmonella species as non-lactose and non-sucrose fermenters that produce H2S form colorless colonies with black centers.

Shigella species (non-lactose and non-sucrose fermenters, no H2S production) form colorless colonies.

Lactose and sucrose fermenters (E. coli, K. pneumoniae) form orange to yellow colonies due to acid production.


Salmonella-Shigella Agar Beef extract 0.5% Peptone 0.5% Bile salts 0.85% Sodium citrate 0.85% Brilliant green dye Trace Lactose 1.0% Sodium thiosulfate 0.85% Ferric citrate 0.1% Neutral red Agar 1.4% pH = 7.4


Salmonella-Shigella (SS) Agar

Bile salts, citrates, and brilliant green dye inhibit gram-positives and most gram-negative coliforms

Lactose the sole carbohydrate Sodium thiosulfate a source of sulfur for

H2S production Salmonella forms transparent colonies with

black centers Shigella forms transparent colonies without

blackening Lactose fermentative Enterobacteriaceae

produce pink to red colonies with bile precipitate for strong lactose fermenters


Use of Selective-Differential Agars for Recovery of the Enterobacteriaceae from Different Types of Specimens

Feces1: MAC or EMB + XLD &/or SS or HE2

Sputum and Urine1: MAC or EMB Wound3:MAC or EMB Peritoneal and pleural fluid4: MAC or EMB Subculture of blood positive for gram-negative’s in

broth culture4: MAC or EMB CSF, pericardial fluid, synovial fluid, bone marrow5:

Not required

1Heavy population of commensal bacteria2Utilized with enrichment broth containing selenite or

mannitol to differentially inhibit enteric commensals3Commensal bacteria (skin) and frequent polymicrobial

etiology 4Possible polymicrobial etiology (normally sterile

fluids)5Normally sterile, unimicrobial etiology predominantwww.drjayeshpatidar.blogspot.in

Selectivity of Differential Agars for Salmonella1 and Shigella2

HE or SS agar (absence of lactose fermentation1,2, H2S production1)

XLD agar (absence of lactose fermentation1,2, H2S production1, lysine decarboxylation1)

MAC or EMB agar (absence of lactose fermentation1,2)

TSI agar (glucose fermentation1,2, absence of lactose fermentation1,2, H2S production1)

Descending Order of Selectivity for Salmonella and Shigella


Recommended Reading

Winn, W., Jr., Allen, S., Janda, W., Koneman, E., Procop, G., Schrenckenberger, P., Woods, G.Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Sixth Edition, Lippincott Williams & Wilkins, 2006: Chapter 5. Medical Bacteriology:

Taxonomy, Morphology, Physiology, and Virulence.

Chapter 6. The Enterobacteriaceae. www.drjayeshpatidar.blogspot.in

Recommended Reading

Murray, P., Baron, E., Jorgensen, J., Landry,

M., Pfaller, M.Manual of Clinical Microbiology, 9th Edition, ASM Press, 2007: Farmer, J.J., III, Boatwright, K.D., and

Janda J.M. Chapter 42. Enterobacteriaceae: Introduction and Identification


Thank You


the enterobacteriaceae basic properties.ppsx x

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