Practical 6: Investigation of Gram Negative anaerobic bacilli

Question 1

List the gram negative anaerobic bacilli
(5)

Answer 1

Bacteroides

Porphyromonas

Prevotella

Fusobacterium

Pasteurella (practical 5)

Question 2

How do you differentiate pasteurella from the bacteroidaceae

Answer 2

Pasteurella is a coccobacilli while the bacteroidaceae are GNBs

Question 3

List the genera that make up the family bacteroidaceae
(4)

Answer 3

Bacteroides

Poryphyromonas

Prevotella

Fusobacterium

Question 4

What are the three clinically significant species of bacteroides?

Answer 4

B. fragilis

B. oralis

B. ureolyticus

Question 5

What % of anaerobe cultures are B.fragilis

Answer 5

25%

Question 6

How can you identify bacteroides

Answer 6

Pleomorphic bacilli or coccobacilli -> different shapes

Non-endospore forming

Question 7

How could you identify a bacteroides from a case study?
(3)

Answer 7

Bacteroides are opportunistic pathogens

May be seen following surgery e.g. bowel surgery

They may be isolated from abscesses and other legions

Question 8

How could you identify B. fragilis from a case study?

Answer 8

Frequently associated with intra-abdominal and soft tissue infections below the waist or bloodstream infections

Question 9

What are the five strains of fusobacterium

Answer 9

F. nucleatum

F. necrophorum

F. periodonticum

F. mortiferum

F. necrophorum

Question 10

How can you identify fusobacterium from the case study
(2)

Answer 10

Commensals of the respiratory tract and GIT

Colonise mucous membranes of humans and animals

Question 11

What fusobacterium strains are found in the oral cavity?
(3)

Answer 11

F. nucleatum

F. necrophorum

F. periodonticum

Question 12

What fucobacterium strains are seen in intra-abdominal infections
(3)

Answer 12

F. nucleatum

F. mortiferum

Usually both seen together

Question 13

What fucobacterium strain causes Persistent Sore Throat Syndrome

Answer 13

F. necrophorum

Question 14

What is the only clinically significant strain of pasteurella

Answer 14

P. multocida

Question 15

How could you identify pasteurella from a case study

Answer 15

A pet bite with cellulitis and bloody drainage at site of wound

Question 16

What is the only clinically significant strain of prevotella?

Answer 16

Pr. melaninogenica

Question 17

How could you identify Pr. melaninogenica from a case study

Answer 17

Indigenous to the oral cavity

Question 18

How do you identify porphyromonas from the ID?

Answer 18

Black colonies

Gram negative bacilli

Question 19

What three tests should be carried out on gram-negative bacilli

Answer 19

Anaerobic growth

Metronidazole susceptibility

Anaerobic identification discs -> MID8

Question 20

What is the principle behind the anaerobic and metronidazole susceptibility test for anaerobic GNBs
(3)

Answer 20

We need to prove that the bacilli are in fact anaerobic

Need to put up an aerobic and anaerobic plate -> the organism must grow on the anaerobic plate

Anaerobic GNBs are susceptible to metronidazole so a disc should be put up on the anaerobic plate to prove its an anaerobic GNB

Question 21

How do you carry out an anaerobic metronidazole susceptibility test for anaerobic GNBs
(4)

Answer 21

Ask for x2 blood agar plates and a metronidazole disc and your controls (Bacteroides fragilis + and pseudomonas fragilis -)

Split your plates into three sections for your controls and test

Inoculate both plates

Add metronidazole discs to your test inoculum

Question 22

What is your positive control (anaerobic growth and metronidazole susceptibility)

Answer 22

Bacteroides fragilis (anaerobic growth only)

Question 23

What is your negative control (non anaerobic/aerobic growth and metronidazole susceptibility)

Answer 23

Pseudomonas aeruginosa (aerobic growth only)

Question 24

What is the MID8 Identification Mastring Test?
(2)

Answer 24

Anaerobic Identification Discs

A commercial system for the identification of gram negative anaerobic bacteria based on the susceptibility to six antimicrobials

Question 25

Why can the MID8 Identification Mastring Test not be used for susceptibility testing of the GNBs

Answer 25

Due to the high concentrations of some of the antibiotics used in the MID8

Question 26

What six antimicrobials make up the MID8 ID Mastring test

Answer 26

Erythomycin

Eifampicin

Colistin sulphate

Penicillin

Kanamycin

Vancomycin

Question 27

How do you carry out an MID8 ID Mastring Test
(6)

Answer 27

Ask for MID8 mastring (list antimicrobials), blood agar and a McFarland 0.5 standard

Great a dense suspension of organism equivalent to a McFarland 0.5 standard

Lawn inoculum

Using a sterile forceps press the MID mastring onto the plate

Incubate the plate anaerobically at 37 degrees for 48 hours

Do the same for your B.fragilis control

Question 28

How should you incubate your MID8 ID mastring test

Answer 28

incubate anaerobically at 37 degrees for 48 hours

Question 29

What is your positive control for MID8 ID mastring test

Answer 29

B. fragilis

Question 30

Why do you use B. fragilis as your positive control for MID8
(6)

Answer 30

Erythromycin Susceptible (occasionally)

Rifampicin susceptible

Colistin Resistant (occasionally)

Penicillin resistant

Kenamycin resistant

Vancomycin resistant