Microbiology Week 3

Which enteric pathogens causes bacillary dysentery?

Shigella

How are these infections acquired?

Fecal/Oral

Contaminated food/water

Human carriers

Which enteric pathogens cause enteric fevers as well as gastroenteritis and septicemia?

Salmonella

Which of these causes typhoid fever?

Salmonella typhi

Which enteric pathogen causes gastroenteritis as well as an infection that mimics appendicitis? What is the name of that infection?

Yersinia enterocolitica, ileitis or mesenteric adenitis

Which pathogen can live at refrigerator temperatures, and thus can cause transfusion sepsis?

Yersinia enterocolitica

Which pathogen causes bubonic plague?

Yersinia pestis

What are three manifestations of the plague?

Pneumonic plague

Bubonic plague

Septicemic plague

How is the causative agent of plague transmitted?

Infected flea bites

Direct contacts with rodents

Pneumonic form is trasmitted person to person

Which enteric is a common cause of pneumonia?

Klebsiella pneuminiae

Which enteric is most commonly isolated in the clinical lab, causing gastroentertis, UTI, meningitis, septicemia, wound infections, and traveler's diarrhea?

E. coli

Which strain of E. coli causes a shigella-like diarrhea along with uremia and has been acquired by eating undercooked hamburger, drinking cider and unpasteurized milk?

Enterohemorrhagic E. coli (EHEC, E. coli O157:H7 is an example)

or Shigella like Toxin E. coli (STEC)

What enteric is the 3rd most common isolate, especially UTI and wounds?

Proteus mirabilis

What enteric is associated with gastroenteritis and wound infections associated with fresh water/aquaria?

Edwardsiella tarda

Normal ranges for CSF

WBCs: 0-5/uL

Glucose: 40-70 mg/dL (60-70% serum value)

Protein: 15-45 md/dL

CSF ranges for bacterial meningitis

WBCs: >1000/uL; segs

Glucose: decreased <40mg/dL

Protein: increased >100mg/dL

A urine specimen is plated with a 10 uL calibrated loop. After 24 hrs, 83 colonies of a lactose negative gnb are noted on MAC. There was no growth on CNA. What is the colony count?

83 x 100 = 8300 cfu/mL

A gram stain screen is performed on a sputum specimen. There are TNTC (too numerous to count) squamous epithelial cells and no WBCs noted on the smear. Should this specimen be accepted? What do the smear results indicate?

No, it should be rejected. It came from the throat and it will be contaminated with too much normal flora

State the most common battery of media for a bloody stool. For each media list/describe colonies of the pathogen(s) each will recover.

A SMAC plate and an HE, XLD, or SS plate.

SMAC - E. coli O157:H7 will be clear and all other E. coli strains will be bright pink

HE: Shigella will be blue-green, E. coli will be bright orange or salmon pink

XLD: shigella will be red or colorless; E. coli will be yellow

State two purposes for including EMB or MAC in the battery of primary isolation media

Inhibits gram positives/Selects for gram negatives

Differentiates based on lactose fermentation

What is the common sugar (CHO) in EMB and MAC?

Lactose

What is the indicator(s) of lactose fermentation in: EMB

Eosin and methylene blue

What is the indicator(s) of lactose fermentation in: MAC

Neutral Red

What are the inhibitors in: EMB

Eosin

Methylene Blue

What are the inhibitors in: MAC

Bile salts

Crystal violet

What color is a lactose negative (non-lactose fermenter) enteric on EMB & MAC

Colorless colonies

What color is a lactose positive (lactose fermenter) enteric on: EMB

Dark purple

What color is a lactose positive (lactose fermenter) enteric on: MAC

Bright pink

Name two commonly isolated enterics that are lactose positive

E. coli

Klebsiella

What would the color (slant/butt) on TSI media of an enteric that produces dark purple colonies on EMB and bright pink colonies on MAC be? Explain

Yellow/yellow

All enterics are glucose fermentors, so the butt will be yellow

Lactose fermenters will produce a yellow slant (we know it's a lactose fermenter because it grew dark purple on EMB and bright pink on MAC)

What would the color (slant/butt) on TSI media of an enteric that produces clear colonies on EMB and MAC be? Explain

Red/yellow

All enterics are glucose fermentors, so the butt will be yellow

Non-lactose fermenters will produce red slant (we know it's a non-lactose fermenter because it grew colorless on EMB and MAC)

Explain principle of red slant on TSI

The red slant is produced because the organism catabolizes peptones in the presence of oxygen, which produces an alkaline product that changes the phenol red indicator back to red

Name three stool PIMs (and their abbreviations) used to screen for Salmonella and Shigella

Helstoen (HE)

Salmonella-Shigella Agar (SS)

Xylose Lysine Deoxycholate (XLD)

What are the primary inhibitors in these media?

Bile salts

What do Salmonella colonies look like on: HE

Blue-green

Black center

What do Salmonella colonies look like on: SS

Colorless

Black center

What do Salmonella colonies look like on: XLD

Red

Black centers

What do Shigella colonies usually look like on this media at 18-24 hours: HE

Colorless or green

What do Shigella colonies usually look like on this media at 18-24 hours: SS

Colorless

(Except S. sonnei which is pink at 48 hours)

What do Shigella colonies usually look like on this media at 18-24 hours: XLD

Colorless or red

What color are Salmonella and most Shigella on EMB & MAC? Why?

Clear

They do not ferment Lactose

Why is SMAC used for stool PIM?

If E. coli O157:H7 is suspected because a stool is bloody

What sugar (CHO) is in SMAC?

Sorbitol

What color are E. coli O157:H7 colonies on SMAC? Why?

Clear

O157:H7 is sorbitol negative; all other E. coli strains do and will be bright pink

What enteric (gut) pathogen does CIN recover?

Yersinia enterocolitica

What is the sugar (CHO) in CIN?

Mannitol

At what temperature is CIN best incubated?

25 degrees C

Describe Y. enterocolitica colonies on CIN.

"Bulls Eye" colonies

Thin, pink halo rim with large, bright pink/red center

What does TSI stand for?

Triple Sugar Iron media

What is it used primarily to detect?

Glucose and lactose (or sucrose) fermentation by gram negative bacilli. It also detects H2S and gas producers.

What is the pH indicator of sugar fermentation?

phenol red

What in the media detects H2S production?

Sodium thiosulfate and iron salts

Describe proper inoculation/incubation of TSI media

1. Inoculation: Using an inoculating needle with the test organism, stab the media within 1/2" from thebottom (butt/deep) of the tube; as the needle is withdrawn, streak the slant.

2. Loosely recap the tube.

3. Incubation: Incubate in aerobic incubator at 350C/24 hrs.

What three sugars are in TSI media?

Glucose

Lactose

Sucrose

What color does the media turn when the sugar is fermented?

Yellow

What is the visual indication in TSI media that H2S has been produced?

Blackening of the media

What is the indicator that gas has been produced in TSI media?

Cracks in the media

What is the ratio of sugars in TSI media?

1 Glucose

10 Lactose

10 Sucrose

Interpret an all red tube

K/K (alkaline/alkaline)

No sugars fermented; must check for growth on slant

Interpret a tube that is red/yellow

K/A (alkaline/acid)

Only glucose fermented

Interpret a tube that's yellow/yellow

A/A (acid/acid)

Ferments glucose and lactose and/or sucrose

How would a tech determine whether lactose or sucrose or both sugars were fermented?

Levine's EMB plate

Explain why a glucose only fermenter produces the reaction K/A, whereas a lactose fermenter produces the A/A reaction. (Hint: Read/consider the ratio of sugars in the media.)

The organism will ferment the small amount of glucose present and the whole tube will turn yellow, then any organisms that cannot ferment lactose or sucrose will catabolize peptones in the presence of oxygen, which produces an alkaline product that changes the phenol red indicator back to red on the slant.

What is the difference between the TSI and KIA sugar compositions?

TSI contains Sucrose, KIA does not

What medias are used as PIM for stool samples?

SBA

CNA

EMB

HE

SMAC

CIN

What 2 stool pathogens are expected based on the PIM used?

Salmonella

Shigella

What would Salmonella and Shigella colonies look like on EMB? Why?

Clear

They don't ferment Lactose

If Salmonella is present, describe the colonies a tech would see on HE

Blue-green

Black center

Why do Salmonella colonies on HE have black centers?

H2S is produced

If SS agar were used instead of HE, what would Salmonella colonies look like?

Colorless colonies

Black center

If XLD were used instead of HE, what would Salmonella colonies look like?

Red

Black centers

Explain why Salmonella colonies appear red on XLD instead of clear.

LDC+

Assume the tech recovers a possible Salmonella from the stool culture, describe the serological screening that would be done to rule in/out Salmonella typhi

1. Add colonies to saline to make a suspension of organism

2. place a drop of organism in each of 3 wells

3. To well 1 add 1 drop of polyvalent antisera, mix

4. To well 2 add 1 drop of Vi antisera, mix

5. To well 3 add 1 drop of Group D antisera, mix

6. Rotate card

If positive with Vi antisera, boil organism suspension 15-30 minutes and repeat testing with all 3 antisera

Name the antigen and antibodies used to serotype Salmonella from a stool, urine or blood culture

Salmonella O (cell wall) antigens and Vi (capsule) antigens, and Anti-O and Anti-Vi

Why might the suspension for serological typing of a possible Salmonella be boiled and retested?

To destroy the capsule to reveal the cell wall (O) antigens

Distinguish between the TSI reactions of Salmonella typhi and other Salmonella species

"mustache" appearance

Weak H2S production

Which Salmonella species may be ONPG positive?

Salmonella arizonae

Are Salmonella species motile or non-motile?

Motile

Describe how the colonies of Shigella would like like on MAC

Clear

Describe what Shigella colonies would look like on HE

Green or clear

If SS agar were used for PIM instead of HE, what would Shigella colonies look like?

Colorless

If XLD were used instead of HE, what would Shigella colonies look like?

Colorless

Why do Shigella colonies appear clear on MAC, EMB, HE, SS, and XLD?

They don't ferment Lactose, sucrose, or xylose

Would Shigella/Salmonella grow on CNA?

No

If Shigella were recovered from a stool culture, describe the serological typing that could be identified

1. Add colonies to saline to make a suspension of organism

2. Place a drop of organism in each of the 4 wells

3. To well 1 add 1 drop of Group A antisera, mix

4. To well 2 add 1 drop of Group B antisera, mix

5. To well 3 add 1 drop of Group C antisera, mix

6. To well 4 add 1 drop of Group D antisera, mix

7. Rotate card

If no reaction with any antisera, boil the organism suspension 15-30 min and repeat testing with all 3 antisera

If Shigella were recovered from a stool culture, describe the species that could be identified

Group A: S. dysenteriae

Group B: S. flexneri

Group C: S. boydi

Group D: S. sonnei

Why might a tech boil a suspension of suspect Shigella organism, and retest it for Shigella?

To remove the heat labile capsule

What kind of antigens are used to type for Shigella?

Somatic O antigens

Describe the TSI reaction of Shigella species

Red/Yellow

K/A

Which Shigella species is ONPG and ODC positive?

S. sonnei

Are Shigella species motile or nonmotile?

Nonmotile

Why was SMAC included in the battery?

Suspect E. coli O157:H7

What does SMAC stand for?

Sorbitol MacConkey

What would E. coli O157:H7 colonies look like on SMAC?

Clear colonies

Explain why the colonies are colorless on SMAC

They do not ferment Sorbitol

Describe the antigen and antibody used to serotype a colony of E. coli O157:H7

Antigens O157 (cell wall) and H7 (flagella)

Anti-O157, Anti-H7

Stool PIM

SBA, CNA, MAC, HE, CIN

What stool pathogen is suspected since CIN was included in the PIM?

Yersinia enteroclitica

At what temperature is CIN incubated? Why?

25 degrees C

Y. enterocolitica grows and ferments Mannitol best at 25 degrees C

Describe the unique motility pattern of Yersinia enterocolitica

Motile when grown below 30 degrees C