Biochemical Identification of BIOCHEMICAL PATHWAYS FROM GLUCOSE TO PYRUVIC ACID

Clinically-Relevant Enterics •starting carbohydrate for bacterial fermentation or oxidation is glucose

•bacteria use other sugars as carbon source, they convert the sugar to glucose, which is processed by one
of three pathways
Bacterial Biochemistry and Metabolism 3 major biochemical pathways bacteria use to breakdown glucose to pyruvic acid are:
1. Embden-Meyerhof-Parnas (EMP) glycolytic pathways
2. Pentose Phosphate pathways
METABOLISM
⮚ Microbial metabolism consists of: 3. Entner-Doudoroff pathway

✔ biochemical reactions bacterial use to breakdown organic compounds ANAEROBIC UTILIZATION OF PYRUVIC ACID FERMENTATION
Some fermentation pathways used by the microbes that inhabit the human body are as follows:
⮚ Energy from the new constructions is generated during the metabolic breakdown of the
• Alcoholic fermentation
substrate
⮚ Metabolism can be regulated in the cell either by regulating: ✔ End product is ethanol
• production of the enzyme – can be induced or suppressed by molecules ✔ Used by yeast when they ferment glucose to produce ethanol
present in the cell
• Homolactic fermentation
• activity of the enzyme – products of the enzymatic reaction or a
succeeding enzymatic reaction inhibit the activity of the enzyme ✔ End product is only one acid, lactic acid
⮚ Bacteria vary widely in their ability to use various compounds as substrates and in the end ✔ All members of the genus Streptococcus and many members of genus
products generated Lactobacillus ferments pyruvate using this pathways
• Heterolactic acid
•Diagnostic schemes analyze each unknown microorganisms for:
✔ Mixed fermentation pathways
1. Utilization of various substrate as carbon source
✔ End products aside from lactic acid, carbon dioxide, alcohols, formic acid and
2. Production of specific end products from various substrates acetic acid
3. Production of an acid or alkaline pH in the test medium • Propionic acid fermentation
✔ Propionic acid is the major end product of fermentations carried out by
FERMENTATION AND RESPIRATION Propionibacterium acnes and some anaerobic non – spore-forming, gram positive
• Bacteria use biochemical pathways to catabolize (break down) carbohydrates and produce bacilli
energy by two mechanisms:
• Mixed acid fermentation
1. Fermentation
✔ More acid end products : lactic, acetic, succinic and formic acid
2. Respiration
✔ Used by members of the genera Escherichia, Salmonella, Shigella
RESPIRATION (Enterobacteriaceae)
• an anaerobic process carried out by both obligate and facultative anaerobes
✔ Basis for positive reaction on the methyl red (MR) test
• less efficient in energy generation than respiration
• Butanediol fermentation
• end products are analyzed for identification of anaerobic bacteria
• Efficient generating process in which molecular oxygen is the final electron acceptor ✔ End products are acetoin (acetyl methyl carbinol) and 2,3-butanediol
• Obligate aerobes and facultative anaerobes carry out aerobic respiration ✔ Detection of acetoin is the basis for positive voges proskauer (VP) test
• Certain anaerobes can carry out anaerobic respiration ✔ Little acid is produced by this pathway
• inorganic forms of oxygen, such as nitrate and sulfate, act as the final electron ✔ Thus, organisms that have a (+) VP reaction usually have (-) reaction on MR and
acceptors vice versa
 AEROBIC UTILIZATION OF PYRUVATE (OXIDATION)

Krebs or Tricarboxylic acid (TCA) cycle

• most important pathway for complete oxidation of a substrate under aerobic conditions ● Important step in classifying members of the family Enterobacteriaceae is the determination
• pyruvate is oxidized of microorganism’s ability to ferment lactose
● These bacteria are classified as:
• results is production of acid and the evolution of carbon dioxide
• LF – possesses both β-galactoside permease and

BIOCHEMICAL TEST β-galactosidase enzymes

• designed to demonstrate the physiological and chemical characteristics of microorganisms • NLF – do not possess either enzyme

• enables the bacteriologist, by elimination, to identify the microorganism specifically • dLF or late LF – lack β-galactoside permease but possess β-galactosidase

CARBOHYDRATE UTILIZATION Other carbohydrates used by bacteria:

• Fermentation of sugar is usually detected by: (ultimately converted into glucose for use in glycolysis)
⮚ acid production • Maltose Raffinose
⮚ change of color resulting from pH indicator present in the medium • Rhamnose Arabinose
• Bacteria generally ferment glucose preferentially over other sugars • Sucrose
⮚ glucose must not be present if the ability to ferment another sugar is being
tested
• most important carbohydrate determination test is determining lactose utilization •Polyhydric alcohols (which end in “ol”, collectively called sugars), include:

• Lactose degradation can be used to differentiate bacterial species as: • Adonitol

⮚ lactose fermenters (LF) • Dulcitol

⮚ non-lactose fermenters (NLF) • Mannitol

• Lactose is a disaccharide consisting of one molecule of glucose and one molecule of • sorbitol
galactose linked together by a galactoside bond
• Two steps involved in the utilization of lactose by a bacterium
• Enzymes necessary for a bacterium to take up OXIDATION-FERMENTATION TEST
lactose and cleave it into monosaccharides
⮚ when fermentation occurs, a mixture of end products accumulates in the medium
1st step requires an enzyme
• production of pyruvic acid which can be broken down further to other acids
β-galactoside permease (lactose permease) forming large amount of acids or acid with gas
• serve as transport enzyme that facilitates entry of the lactose molecules across the • acid formation is detected with pH indicators added in the medium
bacterial cell plasma membranes
⮚ higher acidity is produced during fermentation than during oxidation
2nd step occurs inside the cell wall, requires the enzyme ⮚ oxidation begins by glucose entering the glycolysis pathways
β-galactosidase ⮚ pyruvic acid formed from glycolysis is metabolized further to carbon dioxide (CO2 )
⮚ to break the galactoside bond, releasing glucose and galactose • requires oxygen (aerobic respiration) or another inorganic molecule
⮚ glucose is now available for metabolism and can be fermented primarily (anaerobic respiration), such as nitrate (NO3) as a terminal electron
through the Embden Meyerhof pathway, also referred to as glycolysis acceptor

⮚ Alternatively, bacteria can use the Entner Doudoroff pathway

Hugh and Leifson O/F basal medium (OFBM) Triple Sugar Iron and Kligler’s Iron Agar
• Uninoculated medium is red because the pH is buffered at 7.4
•1% carbohydrates
• Both TSI agar and KIA are useful in detecting the ability of the microorganism:
•0.2% peptones ✔ To ferment carbohydrates, glucose and lactose in KIA and glucose, lactose, or
sucrose in TSI agar
•pH indicator – bromthymol blue
✔ To produce gas from the fermentation of sugars
•acidic environment – yellow ✔ To detect the production of H2S
• Both TSI agar and KIA are poured on a butt/slant – slant portion is aerobic; the butt, or deep
•alkaline environment – blue
portion, is anaerobic
•original color of the medium • To inoculate TSI agar or KIA, the laboratory scientist should pick a well-isolated colony with
an inoculating needle and stab the butt almost all the way to the bottom of the tube
• Laboratory scientist moves the needle back and forth, known as fish tailing, across the surface
of the slant
• The cap is replaced loosely to allow oxygen to enter the tube, and the medium is incubated in
a non-CO2 incubator for 18 to 24 hours
• The reaction patterns are written with the slant results first, followed by the butt reaction,
separated by a slash (slant reaction/butt reaction)
• It is important that the reactions be read within an 18- to 24-hour incubation period;
otherwise, erroneous results are possible

Carbohydrate Fermentation with

Triple Sugar Iron (TSI) and Kligler’s Iron Agar (KIA)
• TSI is a butt/slant medium considered to be a modification of KIA
• TSI and KIA are useful in the presumptive identification of gram-negative enteric bacteria

• Carbohydrate Content
1. TSI
glucose (0.1%)
lactose (1.0%)
sucrose (1.0%)
2. KIA
glucose (0.1%)
lactose (1.0%)
• pH indicator – phenol red (yellow below
pH 6.8)
• H2S indicator - ferrous sulfate and sodium
thiosulfate
Principle -------> ------->

• Glucose utilization by certain organisms occurs both:

• aerobically on the slant where oxygen is available
• anaerobically in the butt
• once glucose is fermented acids will be produced
• acids in the medium will cause the phenol red to assume a yellow color
• butt and slant will appear yellow after 6 hours of incubation
• After depletion of the limited glucose (0.1%), some organisms utilize lactose or sucrose and
continue making acid end products The reaction is called alkaline over acid (K/A) signifying that only glucose was fermented

• slant and butt will remain yellow after 18-24 hours incubation No fermentation: Glucose or lactose non-fermenters (K/K)
• reaction is called acid over acid (A/A), fermenting two or all of the sugars •Glucose non-fermenters may also produce alkaline products from peptone utilization
•Reaction seen will be alkaline over alkaline (K/K)
•signifies that no sugars was fermented
------->

------->

Glucose fermentation only (K/A)

Lactose (or sucrose or both) fermentation (A/A)

•fermentation of 0.1% glucose------> acid production
oGlucose fermenters attack the simple sugar glucose first and then lactose or sucrose
•changes initially the color of the entire medium to yellow oAcid production from the fermentation of additional sugar is sufficient to keep both the slant and butt
•12 hours of incubation, the glucose is consumed and bacteria on the slant utilize peptones aerobically, oit is important that the TSI and KIA tests are not read after 24 hours of incubation
producing an alkaline reaction which changes the indicator to a deep red color
•Fermentation of glucose (anaerobic) in the butt --------> larger amounts of acid, overcoming the
alkaline effects of peptone degradation
•therefore the butt remains acidic (yellow)
•After incubation the TSI will show a red slant and a retained yellow butt
Reactions to TSI and KIA H2S production

H2S production is a two-step process

1st step
● Bacterium (acid environment) + sodium thiosulfate ----> H2S
H2S is a colorless gas

2nd step
● H2S + Ferric ions -----> ferrous sulfide (black ppt.)
● H2S production requires an acid environment

Gas Production

•Gas production (aerogenic) or no gas production (non-aerogenic):

• Production of gas results in either of the following:
✔ formation of bubbles IMViC Reaction
(Indole, Methyl Red, Voges-Proskauer, Citrate)
✔ splitting of the medium in the butt
INDOLE TEST
✔ complete displacement of the medium from the bottom of the tube
Principle
● organism that produce the enzyme tryptophanase are able to degrade tryptophan into indole
Ortho-Nitrophenyl-β-D-Galactopyranoside Test (ONPG) and the p-nitrophenyl-β-D ● indole is detected by its ability to combine with certain aldehyde to form a colored
galactopyranoside (PNPG) compound
• Determines whether the organism is dLF (one that lack the enzyme β-galactoside permease but
possesses β-galactosidase) or a true NLF Kovac’s method

• ONPG is structurally similar to lactose, but ONPG is more readily transported through the • Indole indicator: Kovac’s reagent (p dimethylaminobenzaldehyde or PDAB)
bacterial plasma membrane and does not require β-galactoside permease • Result - development of a pink to deep red color which is a (+)
• β-galactosidase hydrolyzes ONPG, a colorless compound, into galactose and o- nitrophenol, a
yellow compound (+) dLF Erhlich’s Method

• ONPG remains colorless if the organism is a NLF a. Indole indicator:

✔ Erhlich’s reagent (p-dimethylaminobenzaldehyde)
• β-galactosidase is an inducible enzyme
b. Result - development of a brilliant red ring between the solvent
• the gene is expressed only if lactose or a similar substrate is present
and the medium
• therefore, bacteria should be taken from lactose-containing media for testing
 Voges Proskauer
Spot Indole test
• Acids formed during fermentation can be metabolized further to 2,3-butanediol through the
intermediate acetoin
This is a rapid method for the detection of indole.
• α-naphthol – catalyst or color intensifier
a. Indole indicator:
• 40% KOH or NaOH - provide alkalinity
✔ p-dimethylaminocinnamaldehyde
• Tube is gently shaken – to increase oxygenation
b. Result:
• Bacteria tend to be positive for either MR or VP but not both
✔ development of a blue or blue green color

Glucose metabolism and its metabolic products Principle

• Enterics takes two separate pathways for its metabolism:
•Glucose ---> Pyruvic acid ---> Acetoin
• Mixed acid fermentation pathways
----> Diacetyl + KOH + α-naphthol
• Butylene glycol pathways
---->Red complex ---> 2,3-Butanediol
• MR VP tests – detects the end products of glucose fermentation
Methyl Red Test
Result
Medium: MR-VP or Clark and Lubs Positive – red color (pH 4,5 and below)
dextrose broth medium
Negative – yellow color (pH over 4.5)
ph Indicator: methyl red
Principle:

Glucose ----> Pyruvic acid ----> Mixed acid fermentation (pH 4.4) ---->

Red color with methyl red indicator

 UREASE TEST
Citrate Test
Medium: Christensen’s urea agar or urea broth
pH Indicator: phenol red
Medium: Simmon’s citrate slant
Principle:
based upon the ability of some bacteria to
pH indicator: bromthymol blue hydrolyze urea into ammonia, water and CO2 by
means of the enzyme urease
Principle: Results:
Some organisms can utilize citrate as their sole of • Positive – bright pink or red
carbon producing acetate and other alkaline carbonate • Negative – yellow
end products in the process.
These products change the color of the indicator from
green to blue.
Result:
Positive – Prussian blue color (alkaline)
Negative – no color change (green) AMINO ACID UTILIZATION
Decarboxylase and dihydrolase test

• Ability of bacteria to use amino acids as energy and carbon sources

OXIDASE TEST • Decarboxylase test determine whether the bacterial species possess enzymes
• Determines the presence of the cytochrome oxidase system that oxidizes reduced cytochrome capable of decarboxylating (removing the carboxyl group, COOH) specific amino
with molecular oxygen acids in the test medium
• Differentiates Enterobacteriaceae (-) from Pseudomonadaceae (+)
• Useful in identifying Neisseria spp. • Two amino acids commonly used to test for decarboxylase activity:

• Modified oxidase test – differentiates Staphylococcus from Micrococcus • Lysine

• Principle: • Ornithine
• Some organism produce cytochrome oxidase, which in the presence of air, acts on certain • Products of decarboxylation are:
aromatic amines to produce colored compounds.
• Amine or diamine molecules
• Methods:
Spot Oxidase Test (Kovac’s Method) • CO2

a. Reagent: 05% or 1% tetramethyl – p - phenylenediamine

dihydrochloride Degradation of amino acids and their specific products:
• Result:
• lavender is the (+) result • Lysine (amino acid) ----> Lysine decarboxylase ---> Cadaverine (diamine) + CO2

•Ornithine ---> Ornithine decarboxylase ----> Putrescine (diamine)+ CO2

•Cadaverine and putrecine are stable in anaerobic conditions

 Principle:
Arginine can be decarboxylated in two-step process:
• Some organisms can decarboxylate the amino acid lysine to form cadaverine leading to the
alkalinization of the medium and subsequent change in color of the indicator from yellow to
purple
•Arginine ---> arginine decarboxylase ---> agmatine + CO2
• Decarboxylation of lysine occurs anaerobically in the butt
•Agmatine (catabolized further) ---> Putricine + Urea • Deamination of lysine occurs in the slant
• Dark purple butt – positive for lysine decarboxylation
•If the bacteria produce urease, the urea is degraded to ammonia and CO2
• Plum or reddish purple slant – positive for deamination
• H2S production in LIA occurs only in alkaline environment
Arginine can be degraded by arginine dihydrolase
• Therefore, a black precipitate indicating H2S is also positive for
decarboxylation
•Arginine ---> arginine dihydrolase ---> Citrulline + Ammonia +
Inorganic phosphate • Butt turns yellow because of glucose fermentation
Results:

•Citrulline ---> undergoes phosphorolytic ---> cleavage ---> ornithine • K/K – positive decarboxylation without H2S production
• K/A H2S – negative decarboxylation with H2S production
• K/K H2S – positive for decarboxylation with H2S production
LYSINE DECARBOXYLASE TEST • R/Y – negative decarboxylation, positive deamination without H2S production
• Useful in conjunction with TSI in screening stool specimen for the presence of enteric
pathogens, differentiating
- Salmonella spp. = lysine-positive
- Citrobacter spp. = lysine-negative

• Useful in differentiating Proteus, Morganella and Providencia spp. from most other members
of Enterobacteriaceae

• Medium: lysine iron agar (LIA) or Moeller’s medium contains:

• Amino acids lysine

• Glucose
• Ferric ammonium citrate
• Sodium thiosulfate

• pH Indicator: bromcresol purple

 DEAMINASE TEST MOTILITY TEST
• Amino acids can be metabolized by deaminases that remove an amine (NH2) group • Motility can be determined by microscopic examination of bacteria or by observing growth in
a semi-solid medium
• Phenylalanine deaminase (PAD) determines whether an organism possess the enzyme that • Differentiates gram-negative bacteria in the Enterobacteriaceae
deaminates phenylalanine to phenylpyruvic acid • Medium: SIM medium (Sulfide, Indole, Motility) or any semi-solid motility test medium.
- Agar concentration of 0.4% or less to allow for free spread of microorganisms
• Proteus, Morganella and Providencia spp deaminates (attacks the NH2 (amine)group instead
of the carboxyl group) amino acids
• Positive – motile organisms spread out or grow away from the
PHENYLALANINE DEAMINASE TEST line of inoculation.
Medium: phenylalanine agar
- cloudiness spreading from the inoculation line

Reagent: 10% aqueous ferric chloride (FeCl3)

Principle:
Organisms like Proteus and Providencia produce phenylalanine deaminase that breaks
down phenylalanine to phenylpyruvic-acid.
Phenylpyruvic acid reacts with the ferric ion to produce a green color.
Positive Result:
• Dark green color on the surface of the slant and in the fluid on the slant.

• Negative – non-motile organisms grow only along the stab

line.
 LIQUEFACTION OF GELATIN
• Differentiates Aerobacter from the members of Klebsiellae
SULFIDE
• H2S is indicated by black precipitate • Principle:

INDOLE ✔ Use to determine the ability of an organism to produce proteolytic enzymes

(gelatinase) that breaks down gelatin into amino acids
• Pink to red color after the addition of Kovac’s reagent is positive for indole
• Gelatinase activity is detected by loss of gelling of gelatin (liquefaction)
• Gelatinase activity if affected by several factors:
✔ Size of the innoculum
✔ Incubation temperature

MALONATE UTILIZATION
• Determines whether the organism is capable of using malonate as its sole carbon source
• pH indicator – bromthymol blue
• Positive result is due to the increased alkalinity from utilization of ammonium sulfate
• Green to blue color of the medium
• Principle:
✔ Bacteria that able to use malonate as sole carbon source also use ammonium
sulfate as a nitrogen source
NITRATE REDUCTION TEST
•Determines whether an organism has the ability to reduce nitrate to nitrite and reduce nitrite further to
nitrogen gas (N2) MANUAL MULTITEST SYSTEMS
Principles of commercial ID systems falls into one of these five categories or a combination:
Medium:
✔peptone broth with KNO3 ✔ pH based reactions
✔there is also an agar test and spot test A using filter paper disks
✔ Enzyme based reactions
Principle:
✔ Utilization of carbon source
• Some organisms possess nitro reductase that can reduce nitrate to nitrite.
✔ Visual detection of bacterial growth
✔ Nitrite combines with addition of sulfanilamide and n-naphthylenediamine
substrate forming a red – colored end product. ✔ Detection of volatile or non – volatile fatty acids by gas chromatography
• However, if the organism further reduces nitrite to nitrogen gas, the test for nitrite will ✔ ID of bacteria can be facilitated by the use of automated or packaged kit systems
yield a negative (colorless) result.
✔ Numeric codes – generated based on the metabolic profiles of each organism
• An additional test must be performed to validate this colorless result for the presence of
unreacted nitrate. ✔ Profile number – represents the identifying phenotype of specific organisms
✔ Metallic zinc catalyzes the reduction of nitrate to nitrite, thus, with the addition of
zinc a negative test will yield a red color, indicating the presence of unreacted
nitrate.

Results:
Positive – red
Negative – no color change
 RAPID AND AUTOMATED IDENTIFICATION SYSTEM
• Clinical outcome of rapid and accurate reporting of results affect patient in two ways:
✔ Early diagnosis
✔ Subsequent selection of appropriate antimicrobial therapy
• Involves commercially packaged identification kits or fully automated instruments
• Kits are miniaturized test systems
• Employed chromogenic or fluorogenic substrates to asses preformed enzymes

AUTOMATED IDENTIFICATION SYSTEM

Principles used:

✔ Turbidity
✔ Colorimetry
✔ Fluorescent assay
⮚ Fully automated systems incubate and read the reactions
⮚ Computer software interprets the result and provide ID
Advantages:
⮚ decreased turnaround time for reporting of results
⮚ statistical prediction of correct ID
⮚ increased data acquisition and epidemiologic analysis
⮚ automated standardization of ID profiles that can reduce analytical errors