Microbiology 1 Unit 3

Front 1

Enterobacteriaceae

Back 1

- Gram (-) rods
- Glucose-fermenting
- Oxidase (-)
- Catalase (+)
- Motile or non-motile
- Non-sporeformers
- Nitrate-reducing (nitrate → nitrite)
- Heat stable lipopolysaccharide (LPS) (doesn't denature by heat)

Front 2

Indole test

Back 2

The amino acid tryptophan can be broken down by enzyme tryptophanase to form indole, pyruvic acid and ammonia as end products. Tryptophanase differentiates indole-positive enterics, such as E. coli and P.vulgaris from indole-negative enterics, such as S. marcescens.

*Media and Reagents:*
SIM with tryptophan and Kovac's reagent.

*Expected Results:*
Positive test : Kovac's reagent combines with indole and turns the surface red.
Negative test: No red color or copper color development

Front 3

Voges-Proskauer Test (IMViC)

Back 3

It is used to identify enteric bacteria based on their pattern of glucose metabolism. The enterics that produce neutral end-products, such as acetoin are detected by VP test.

*Media and Reagent:*
MR-VP medium and Barritt's Reagent A (contains alpha-naphthol) and Barritt's Reagent B (contains KOH).

*Expected results:*
Positive test: acetoin + alpha-naphthol + KOH = red color
Negative test: alpha-naphthol +KOH = copper color

Front 4

Citrate Utilization (IMViC)

Back 4

Citrate is an organic molecule that can be utilized by bacteria that produce the enzyme citrase. Citrase is produced by some bacteria such as E. aerogenes but not by others like E. Coli

*Media and Reagent:*
Simmon's Citrate Agar. It has citrase as the only carbon source and PH indicator bromothymol blue

*Expected results:*
Positive test: Growth and color changes to blue
Negative test: No growth and color remains green

Front 5

oxidase test

Back 5

This test is used to identify microorganisms containing the enzyme cytochrome oxidase (important in the electron transport chain).
It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae.

Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces it to water. In the oxidase test, artificial electron donors and acceptors are provided. When the electron donor is oxidized by cytochrome oxidase it turns a dark purple.

*Expected Results:*
This is considered a positive result. In the picture above the organism on the right (Pseudomonas aeruginosa) is oxidase positive.

Front 6

Mac Results

Back 6

o gram-negative Enterobacteria Escherichia coli and Enterobacter aerogenes ferment lactose
o E. coli produces pink to red colonies often with a reddish bile precipitate surrounding colonies on MacConkey's agar
o E. aerogenes produces pink to red mucoid colonies on MacConkey's agar
o gram-negative bacteria Proteus vulgaris and Salmonella typhimurium grow on MacConkey's agar, but do not ferment lactose (media appears yellow to light pink in color & colonies are colorless; swarming of Proteus is inhibited

Front 7

Hydrogen Sulfide Test (H2S) (IMViC)

Back 7

(left is negative for H2S and right is positive for H2s)

Bacteria use enzyme cysteine desulfurase to hydrolyze the amino acid cysteine, forming hydrogen sulfide as end-product.

*Media and Reagent:*
SIM with cysteine and ferrous sulfate (detects H2S)

*Expected Results: *
Positive Test: H2S production = Black
Negative Test: No H2S production = No blackening of medium

Front 8

Motility

Back 8

This is not a biochemical test, but it can distinguish bacteria. It determines presence of flagella.

*Media and reagent:*
Deep agar

*Expected results:*
Positive test: Growth spread away from the line of inoculation = motile
Negative test: Growth only occurred at the line of inoculation = Non-motile

Front 9

Escherichia coli

Back 9

+ + - -
Gram negative
BA Growth Beta
Mac LF
XLD LF
HE LF
Motility pos
Citrate neg
urea neg
**selective Colorless (NEG) Mac Sorbitol for EHEC.
Serotyping O, H Shiga toxin for EHEC
MacConkey- reddish growth

Front 10

Enterobacteriaceae Overt Pathogens

Back 10

Typhoid fever = Salmonella
- Dysentery = Shigella
- Bubonic plague = Yersinia pestis

Front 11

Pseudomonas aeruginosa

Back 11

Gram Stain Negative - small, clustered bacillus
aerobic
oxidase POS
BA growth
MacConkey Plate- NFL, Yellow growth
XLD NFL
HE NFL
Motility Pos
Citraite Pos
Urea Variable
SPREADING metallic sheen pigment
Odor is fruity grape smell
Growth at 42 degree celcius

Front 12

Proteus vulgaris

Back 12

+ +- -
Motility- pos
Citrate neg
Urea pos
oxidase neg
BA growth
Mac NLF
XLD NLF H2S
HE NLF H2s
Gram- negative
** H2S producer, ecoli is not**
Temp- incubator showed small growth, should grow around 23 degrees

Front 13

Enterobacter cloacae

Back 13

- - + +
Gram negative
oxidase neg
BA=Growth
mac =LF
He =LF
Motility neg
citrate pos
Urea neg


Bact. O2- obligate aerobe

Front 14

Acinetobacter baumannii

Back 14

- - Gram (-) cocobacilli
- Non-fermenting
- Saccharolytic (glucose oxidizer), non-hemolytic**
- Most frequent Acinetobacter isolate**
- Responsible for most nosocomial infections
- Respiratory infections, pneumonia
- Increasing resistance

Front 15

Enterobacteriaceae Opportunistic pathogens

Back 15

Urinary tract infection = E. coli, Enterobacter, Proteus,
- Diarrhea, dysentery = Shigella, EIEC
- Nosocomial infections = E. coli, Enterobacter, Proteus, Serratia

Front 16

Escherichia coli

Back 16

+ + - -
Gram (-) rods
- Glucose fermenter
- Oxidase (-)
- Indole (+) **
- MR (+)
- VP (-)
- Citrate (-)
- β- hemolysis **
- Motile, pos

Front 17

Escherichia coli Clinical Disease

Back 17

Normal bowel flora (~10% of intestinal flora)
- Most common Gram (-) rod in sepsis
- Cause of 80% of community-acquired UTI
- Gastroenteritis in developing countries (poor hygiene)
- Most infections are endogenous (deep tissue)

- Urinary tract infection (most strains can cause this)
- Specific uropathogenic strains, adhesins
- Septicemia
- Neonatal meningitis:

Front 18

Enterohemorrhagic (EHEC)

Back 18

Virulence factor: toxin similar to Shiga toxin which disrupt protein synthesis and A/E lesions that destroy intestinal microvillus leading to decreased absorption
- Disease: Initial watery diarrhea followed by grossly bloody diarrhea (hemorrhagic colitis) with abdominal cramps; little or no fever; may progress to hemolytic uremic syndrome (HUS) (kidneys get knocked out)
- Large intestine
- Jack-In-the-Box: uncooked hamburgers
- Serotype 0157:H7= NEg Mac Sorbitol, OH ag, shiga toxin

Front 19

Salmonella

Back 19

Widely disseminated in nature and associated with various animals
- Animal-to-animal, animal-to-human
- Animal pathogens, human pathogens, animal-human pathogens
- Contaminated food, fecal-oral spread
- S. typhi is only found in humans at times of infection

Front 20

Salmonella Gastroenteritis

Back 20

Diarrhea caused by a wide variety of serotypes that produce infections limited to the mucosa and submucosa of the GI tract
- 6-48 hrs post ingestion, 2-7days duration
- Nausea, vomiting, non-bloody diarrhea**, fever, cramps, myalgia, headache
Characterized by prolonged fever and multisystem involvement, including lymph nodes, liver and spleen.
- Typhoid fever: S. typhi
- Paratyphoid fever: other salmonellae
- Transported to liver, spleen, bone marrow
- 10-14 days post-ingestion: rising fever, myalias, headache, malaise, anorexia → gastrointestinal symptoms

Front 21

Salmonella

Back 21

- + - v
Oxidase neg
Growth on BA
NLF on Mac
NLF h2s on XLD
NLF h2s on HE
Motility Pos
Citrate Pos
Urea NEg
Selective agar SS Tetrath Selenite
Serotyping OKH ag (ABC1,C2, D,E, Vi)

Front 22

Shigella Epidemiology

Back 22

Humans and other large primates are only reservoirs
- Found in humans only at time of infection
- Not part of normal bowel flora
- Person-to-person spread by fecal-oral route by ingestion of contaminated food, water
- Pediatric disease, low personal hygiene

Front 23

Shigella Clinical Diseases

Back 23

Shigellosis, bacillary dysentary (acute inflammatory colitis)
- 1-3 days post ingestion
- Watery diarrhea in S. sonneir (due to Shiga toxin)
- Lower abdominal cramps, tenesmus, pus & blood in stool
- Self-limiting but treat to prevent spread
- Carrier state possible

Front 24

Yersinia General characteristics

Back 24

Smaller, slower-growing rods
- Motile at room temp, non-motile at 37°C **
- Bipolar staining in direct Giemsa stains

Front 25

Yersinia pestis Epidemiology

Back 25

Carried by urban/domestic rats and wild rodents such as squirrel and prairie dog
- Is the only Enterobacteriacea spp that is transmitted from animals by the bite of an insect vector (flea)

Front 26

Yersinia enterocolitica Epidemiology

Back 26

Carried by dogs, cats, rodents, rabbits, pig, sheep
- Not part of normal flora
- Only inhabit the bowel at the time they are causing infection due to contaminated meat, milk, water
- Enterocolitis in cooler regions
- Not common

Front 27

Bubonic plague

Back 27

Yersinia pestis
- Flea
- Characterized by high fever and painful inflammatory swelling of axilla and groin lymph nodes
- Infection rapidly progresses to fulminant bacteremia and lead to pneumonia that is often fatal if untreated

Front 28

Pneumonic plague

Back 28

Yersinia pestis
- Involves the lungs
- Characterized by malaise and pulmonary signs
- The respiratory infection can occur as consequence of bacteremic spread assoc with bubonic plague
- Can also be acquired by the airborne route during close contact with infected victims (Aerosols)
- Rapidly fatal

Front 29

Proteus mirabilis

Back 29

Normal human GI tract flora
- 2nd most common cause of UTI
- Swarmer (waves)

Front 30

Enterobacter, Citrobacter, Morganella, Serratia

Back 30

Normal human GI tract flora
- immunocompromised or nosocomial infections

Front 31

Klebsiella Pneumoniae

Back 31

Normal human GI tract flora
- Nonmotile rod-shaped gram (-) enterobacteria
- Prominent capsule
- K. pneumoniae,

Front 32

Enterobacteriaceae Culture Media

Back 32

Most grow well on SBA, BA, and MAC
- Non-fastidious
- Selective/differential media
- XLD, HE, SS (Salmonella-Shigella agar), CIN agars

Front 33

Enterobacter Media & Colony Appearance

Back 33

MAC: Lactose fermenter; may be mucoid
- HE: yellow
- XLD: yellow

Front 34

E. Coli Media & Colony Appearance

Back 34

MAC: Lac fermenter; flat, pink colonies with surrounding darker pink area of precipitated bile salts
- HE: Yellow
- XLD: Yellow

Front 35

Proteus Media & Colony Appearance

Back 35

MAC: Non lac fermenter; swarming; foul smell
- HE: Colorless
- XLD: Yellow or colorless, with black centers

Front 36

Salmonella Media & Colony Appearance

Back 36

MAC: Non lac fermenter
- HE: Green
- XLD: Red with black center (due to H2S production)

Front 37

Shigella Media & Colony Appearance

Back 37

MAC: Non lac fermenter
- HE: blue-Green
- XLD: Colorless

Front 38

E.coli

Back 38

-a coliform i.e. ferments lactose
-gram -ve rod
-cat +ve, oxi -ve
-facultative anaerobes
-IMViC ++--
-motile
-Urease and H2S -ve
-Lysine decarboxylase +ve

Front 39

Salmonella

Back 39

-a non-coliform i.e. does not ferment lactose
-gram -ve rod
-cat +ve, oxi -ve
-facultative anaerobes
-IMViC -+-+
-motile
-Urease -ve
-H2S and lysine decarboxylase +ve
-need sal-specifc agar and sal-selective broth - would be over-grown by other bact on normal plate
-black centered colonies on XLD agar

Front 40

Klebsiella IMViC

Back 40

--++

Front 41

What is the theory of the TSI test?

Back 41

Term



What is the theory of the TSI test?


Definition



TSI is a differential medium for Gram-negative enteric organisms. It differentiates organisms based on their ability to ferment the lactose and or sucrose present in the media. (Note: all Gram-negative enteric organisms will ferment the glucose.) The TSI media contains disaccharides lactose and sucrose in concentrations of 1% and glucose at a concentration of 0.1%. The lower concentration of glucose allows for the detection of utilization of this substrate alone. Since glucose is a monosaccharide, it will be used first. The small amount of acid produced in the slant of the tube during glucose fermentation is oxidized rapidly, causing the medium to remain red or revert to an alkaline pH. In contrast, the acid reaction (yellow) is maintained in the butt of the tube because it is under lower oxygen tension. After the depletion of the limited glucose, organisms able to do so will begin to utilize the lactose and or sucrose resulting in an acid slant and butt. H2S production (indicated by the blackening in the butt of the tube) is detected by the sodium thiosulfate in the media, which is reduced by some organisms to hydrogen sulfide, which then reacts with ferrous sulfate in the media yielding the typical black iron sulfide. Cracked or elevated media is the result of production of CO2 from the fermentation of sugars.

Front 42

Salmonella IMViC

Back 42

-+-+

Front 43

How is media inoculated for the TSI test?

Back 43

stab the butt of the tube and streak the surface of the slant

Front 44

How long do you incubate the TSI test?

Back 44

18-24 hours. Incubate no longer than 24 hours because of the acid reaction in the slant may revert to an alkaline reaction. This is called reversion.

Front 45

How do you interpret the results of the TSI test?

Back 45

Red slant/yellow butt (K/A) = glucose fermentation only
Yellow Slant/yellow butt (A/A) = glucose, lactose and/or sucrose fermentation
Red slant/red butt (K/NC) = no carbohydrate fermentation
hydrogen sulfide production results in a black precipitate in the butt.
CO2 production is indicated by cracking or elevation of the media.
NOTE: some hydrogen sulfide producing organisms may produce so much of the black precipitate, ferrous sulfide, that the acidity produced in the butt is completely masked. However, H2S is reduces, an acid condition does exist in the butt even if not observable and should be recoded as such.

Front 46

Explain the purpose of the phenol red in the medium for the TSI test.

Back 46

To detect the fermentation of carbohydrates by change of the medium from an orange-red to a yellow color.

Front 47

3.

Acinetobacter Colony & Appearance

Back 47

- BA: Smooth, opaque, raised, creamy, smaller than Enterobacteriacea spp
- MAC: Non lac fermenter, but colonies exhibit purplish/pink hue that may be mistaken for a lac fermenter

Front 48

Acinetobacter Pathogenesis

Back 48

- Clinical isolates are often colonizers
- True infections are usually nosocomial
- Occur during warm seasons
- Most commonly involve GI tract, respiratory tract, wounds, soft tissue, and bacteremia
- Community-acquired infection: pneumonia & wound infection
- Outbreak among soldiers in Iraq: had A. baumannii.

Front 49

Acinetobacter Epidemiology

Back 49

Widely distributed in natural and hospital environments
- Dry surfaces, food, human skin
- May become apart of respiratory flora of pts hospitalized for prolonged periods (ICU, surgery)
- As many as 25% of healthy adults harbor these bacteria on skin, gastrointestinal tract
- Medical instrumentation introduces organism to normally sterile sites

Front 50

Stenotrophomonas General Characteristics

Back 50

Gram (-) rods
- Oxidase (-)
- Oxidation neg - Dnase (+)
- - MAC (+)