Haemophilus influenzae: Antibiotics Sensitivity Testing (AST) in Chocolate Agar
Haemophilus influenzae AST in Chocolate agar
The above image is showing antibiotics sensitivity testing of Haemophilus influenzae and the following are the drugs-
Ampicillin
Ciprofloxacin
Ofloxacin
Co-cotrimoxazole
Ceftriaxone
Cefepime
Chloramphenicol
Meropenem and
Imipenem.
Protocol for testing fastidious organisms, Haemophilus species
For Haemophilus influenzae and H. parainfluenzae, Haemophilus test medium (HTM) is recommended for susceptibility testing. Mueller-Hinton chocolate agar is not appropriate for antimicrobial susceptibility testing of Haemophilus species ( as shown above image). The test method is Kirby Bauer and the direct colony suspension method with the following exceptions:
Inoculum is prepared by suspending pure culture grown on chocolate agar for 20-24 hours in saline or MHB to have the turbidity of 0.5 McFarland standard.
Take the antimicrobial disc (according to the CLSI criteria) from the cartridge by using sterile forceps. Place not more than five antimicrobial discs onto the surface of the agar plate (diameter of 100 mm) or no more than 12 disks on a 150 mm plate.
Incubate at 35°C in 5% CO2 atmosphere or in the candle jar for 16-18 hours.
Measure the diameter of the zone of inhibition and interpret by comparing the breakpoints with CLSI Tables.
Record the zone size of the test and control strains.
Table: Zone diameter interpretative standards for Haemophilus influenzae (in mm)-
Most of the ampicillin resistance among Haemophilus influenzae is caused by the presence of a β-lactamase. In some settings, less than 1% of the clinical isolates of Haemophilus influenzae are ampicillin-resistant due to another mechanism (altered penicillin-binding proteins). For this reason, a negative β lactamase test can be reported as susceptible to ampicillin (without performing the disc test). Since many other antimicrobial agents have a predictable activity against Haemophilus influenzae (3rd generation cephalosporins, quinolones, amoxicillin-clavulanic acid) there rarely exists a clinical need for performing more than a rapid test for β-lactamase. Susceptibility testing, however, remains a useful tool in surveillance programs.
Common media for antibiotics sensitivity testing are- √ Mueller-Hinton agar (MHA): It is for rapidly growing aerobic pathogens. e.g. Staphylococcus species, Enterococcus species, Salmonella species, other Enterobacteriaceae, Vibrionaceae, Pseudomonas aeruginosa,Acinetobacter species, Burkholderia cepacia and Stenotrophomonas maltophilia. √MHA+ 5% sheep blood agar (MHB): It is recommended for susceptibility testing of fastidious organisms like S. pneumoniae and other streptococci. √ Gonococcus (GC) agar + 1% defined growth supplement: It is for susceptibility testing of N. gonorrhoeae. Cysteine–free growth supplement is not useful for disc diffusion test and chocolate agar enriched with other supplements is not appropriate for susceptibility testing of Neisseria gonorrhoeae √ Haemophilus test medium (HTM): HTM is recommended for susceptibility testing of H. influenzaeand H. parainfluenzae.
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Haemophilus Features
Colony characteristics
Colonies are Smooth, low convex, grayish, translucent, about 0.5-1.0 mm in size. The encapsulated strains usually produce larger and mucoid colonies and are about 1-3 mm in diameter.
Odor
Haemophilus species smell may be either bleach or seminal or dirty socks.
Oxidase test
As you know, Haemophilus species are oxidase test positive and therefore test should be positive.
Gram Stain findings
Gram-negative bacilli or gram-negative coccobacilli and sometimes filamentous may be observed.
Use of X, V and XV disks for Haemophilus species identification
Haemophilus spp. have varying requirements for X, V, and XV growth factors. Consequently, the significant differences in growth factor requirements of Haemophilus spp. allows for their differentiation. Differentiation is based on the presence or absence of growth around and/or between disks impregnated with factors X, V, and XV.
Note: Growth around XV disks but no growth at all around X and V disks alone while maybe in between X and V disks is normally suggestive for Haemophilus influenzae as shown below-
# Chocolate agar with bacitracin for screening Haemophilus ||Use of 10U bacitracin: Bacitracin is a polypeptide antibiotic derived from Bacillus subtilis that functions to block cell wall formation by interfering with the dephosphorylation of the lipid compound that carries peptidoglycans to the growing microbial cell wall. Haemophilusis resistant to bacitracin (10U) whereas most common bacteria are sensitive. It makes it easier to screen Haemophilus influenzae in sputum growing around the bacitracin disc as shown below-
#Haemophilus influnezae on Gram stain as shown below-
#Haemophilus influnezae satellite test: Positive as shown below-
# Variety of Haemophilus species identification on basis of X, V, XV, factors, blood agar, and Xylose test as shown below-
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