Micro - Unit 2: Enterobacterales

What order was created after comprehensive genomic analysis of the original family Enterobacteriaceae?

Enterobacterales

What are the seven families of the order Enterobacterales mentioned in the text?

Enterobacteriaceae, Hafniaceae, Morganellaceae, Yersiniaceae, Erwiniaceae, and more

Where are Enterobacterales found?

Ubiquitous:

soil and water

GI tract of mammals

What types of infectious diseases are Enterobacterales incriminated in?

Almost any type of infectious disease

Diarrhea, pneumonia, wound infections, abscess

What is endotoxic shock?

A lethal manifestation of Gram-negative rod infection

What part of LPS causes endotoxin activity?

Lipid A portion of LPS

What are the effects of endotoxin activity?

Changes in blood pressure, clotting, body temp, circulating blood cells, metabolism, humoral & cellular immunity, resistance to infection

What happens 30 minutes after exposure to endotoxin?

Severe hypotension

What happens 1 hour after exposure to endotoxin?

Increase in temperature

What are the three parts of LPS within the Gram-negative cell wall?

1. Outer variable CHO portions = 'O' antigen

2. Core polysaccharide

3. Lipid A circulates in blood

What is the 'O' antigen of LPS?

Outer variable CHO portions

What is the toxic portion of LPS?

LIPID A

What are the general characteristics of Enterobacterales?

GNR, GNCB, plump, round ends, bipolar stain

What type of metabolism do Enterobacterales have?

Facultative anaerobe

What is the catalase test result for Enterobacterales?

Positive but not a test of choice for ID

What is the method of choice for ID of Enterobacterales?

relies upon byproducts of metabolism under aerobic atmosphere

What is the growth appearance of Enterobacterales on SBAP?

Large dull gray

What is the media of choice for spot tests of Enterobacterales?

SBAP

What are the colony appearances of Enterobacterales on SBAP?

Indistinctive colonies unless dry, mucoid, swarming

What do all Enterobacterales ferment?

Glucose

All glucose fermentera

What pathway do Enterobacterales use to ferment glucose?

Embden Meyerhoff pathway

What is the visible reaction in glucose fermentation?

Substrate + indicator = visible rxn

What are the byproducts of glucose metabolism tested in Enterobacterales?

Gas and pH change

What test detects end products of glucose fermentation?

MR/VP test

What is the result of nitrate reduction in Enterobacterales?

Reduce nitrate to nitrite

What are the reagents used in the nitrate reduction test?

1. Alpha naphthylamine

2. sulfanilic acid

3. zinc dust

What enzyme do bacteria contain to reduce nitrate to nitrite?

Nitrate reductase

What is the result of nitrate reduction test if red in 1-2 minutes?

Positive = Enterobacterales Red +

What is the result of nitrate reduction test if NC?

If NC, add zinc dust

NC = negative in 5-10 minutes

What does a urine dipstick nitrite indicator detect?

UTI caused by Enterobacteriaceae

What is the oxidase test result for all members of Enterobacterales?

Negative

exception Plesiomonas

What enzyme does the oxidase test determine?

Intracellular enzyme oxidase - part of electron transport system

What is the reagent for the oxidase test?

Tetramethylpara-phenylene diamine dihydrochloride

What is a positive oxidase test result?

Blue/purple immediately

disregard colour development after 30 seconds

what reagents are added to oxidase test?

1. plate growth

2. filter paper or disc*

3. cotton swab

What causes false positive oxidase test?

Nichrome or stainless steel loops

What are O antigens?

Cell wall antigens, heat stable, may be associated with LPS of cell membrane for GN organisms

What are H antigens?

Protein, flagellar, heat labile

What are K, Vi antigens?

CHO, capsule, heat labile, may mask O and H

What is the purpose of antigen determination?

Assists in study of epidemiology & ID

ex. E. coli O157 H7

Vi = + Salmonella typhi

What are Enterobacterales commonly referred to as?

Enterics, coliforms

60 genera

250 species

What is the old nomenclature for Enterobacterales?

Family Enterobacteriaceae

7 tribes: E. coli, Edwardsielleae, Salmonelleae, Citrobacteriaceae, Klebsielleae, Proteeae, Yersinieae

What is the new nomenclature for Enterobacterales?

Order Enterobacterales families 9

Enterobacteriaceae, Hafniaceae, Morganellaceae, Yersiniaceae, Pantoea, and more

Whats the cultivation media of enterobacterales?

1 SBAP: big gray - Used for spot/rapid testing

2 Enteric agars: lactose + indicator

- MAC (LF or NLF): selective - bile salts + crystal violets, differential - lactose

- HE (F or NF and H2S +/neg) - selective - bile salts, differential - H2S, sugar

What is the appearance of lactose fermenters on MacConkey agar?

Pink/red colonies

What is the appearance of non-lactose fermenters on MacConkey agar?

Colorless colonies

What is the appearance of lactose fermenters on Hektoen agar?

Orange/yellow colonies

What is the appearance of non-lactose fermenters on Hektoen agar?

Green colonies

What does H2S production look like on Hektoen agar?

Black colonies

What are the selective ingredients in enteric media?

Bile salts, dyes (crystal violet, methylene blue)

What is the purpose of bile salts in selective differential media?

Love levels inhibit GP organisms

High levels inhibit GN enterics

What is the purpose of dyes in selective differential media?

Inhibit Gram-positive organisms

What does EMB stand for?

Eosin methylene blue

What does MAC stand for?

MacConkey

What does SS stand for?

Salmonella Shigella

What does XLD stand for?

Xylose lysine desoxycholate

What is the purpose of enrichment broths?

Inoculated with sample, incubated before subculturing

What makes enrichment broth useful?

useful in evaluation of fecal culture where normal flora inhibits recovery of pathogens

Normal flora is inhibited in lAg phase growth

Pathogens encouraged to lOg phase growth

What happens to normal flora in enrichment broth?

Inhibited, in lag phase growth

What happens to pathogens in enrichment broth?

Encouraged to log phase growth

For carbohydrate utilization - What is fermentation ?

Anaerobic metabolic process where bacteria use glucose or other CHO ‘sugar’

Plate media fermenter or NF = a screen

Tube media = a specific test

What is the basal medium for carbohydrate utilization?

Basal medium + CHO + pH indicator

ex. MAC

basal medium = growth, CHO fermented = lactose, pH indicator = neutral red

What is lactose?

Glucose and galactose

• utilization depends on 2 enzymes for carbohydrate utilization

What are the two enzymes required for lactose fermentation?

Permease and beta galactosidase

What does permease do?

Brings lactose into cell

What does beta galactosidase do?

Cleaves lactose inside cell

What test detects beta galactosidase enzyme?

ONPG test

What is the ONPG test result for Escherichia coli?

Positive

What is the ONPG test result for Proteus vulgaris?

Negative

When is biochemical testing performed?

On fresh isolates

How long is biochemical tube testing incubated?

18-24 hours

How long do automated methods take for biochemical testing?

2-4 hours

How do you inoculate a broth?

Inoculate

How do you inoculate a slant?

Streak/ski the slope

How do you inoculate a semisolid?

Straight stab it

What is added for anaerobic metabolism in biochemical tubes?

Oil overlay

How do you inoculate a plate?

Streak plate with a loop

How do you inoculate a KB plate?

Swab

What is the indole test?

Tryptophan is degraded into indole, pyruvic acid and ammonia

What enzyme is required for indole production?

Tryptophanase

What is the spot method for indole test (rapid screen test)?

Filter paper + reagent + isolate, look for blue colour within 1-3 min

What is the reagent for indole spot method?

Dimethyl amino cinnamaldehyde

What is the tube method for indole test?

Inoculate tryptophane broth, incubate overnight, add 5 drops of Kovacs reagent

What is the Kovacs reagent?

para-dimethylaminobenzaldehyde

What is a positive indole test result?

Pink

What is a negative indole test result?

No change (yellow of reagent)

What are other indole reagents?

Ehrlich's (xylene is added), James

What is the citrate test?

Used as sole carbon source for metabolism + ammonium salts

What is the indicator in citrate test?

Bromtohymol blue indicator

What is a positive citrate test result?

Any shade of blue

What is a negative citrate test result?

Green or no change, no growth

Why is a light inoculum used in citrate test?

Dead organisms are a C source

What does IMViC stand for?

Indole, MR - Methyl Red, VP - Voges-Proskauer, Citrate

What does MRVP detect?

Detects end products of glucose fermentation

Glucose metabolized:

Embden-Meyerhoff pathway - produces pyruvic acid

What are the two pathways Enterics degrade pyruvic acid?

• Mixed acid fermentation pathway = MR,

• Butylene glycol pathway = VP

What is the MR (methyl red) test?

Glucose based broth (MRVP broth)

What happens if glucose is metabolized via mixed acid fermentation pathway (MR test)?

Red colour after addition of MR (pH 4.4)

Glucose → pyruvic acid → mixed acids (red colour)

What is VP test used for?

converting acetoin (acethyl methyl carbinol) to diacetyl

What are the reagents for VP test?

0.6 mL 5% alpha naphthol (catalyst)

0.2 mL 40% KOH (oxidizing agent)

What is required for VP test?

Presence of O2, mix tubes vigorously