Haemophilus influenzae: Antibiotics Sensitivity Testing (AST) in Chocolate Agar

Haemophilus influenzae antibiotic sensitivity testing 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:

  1. 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.
  2.  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.
  3.  Incubate at 35°C in 5% CO atmosphere or in the candle jar for 16-18 hours.
  4. Measure the diameter of the zone of inhibition and interpret by comparing the breakpoints with CLSI Tables.
  5. Record the zone size of the test and control strains.

Table: Zone diameter interpretative standards for Haemophilus influenzae (in mm)-

Antimicrobial agent  Resistant  Intermediate  Susceptible

  • Ampicillin 10 µg         ≤ 18             19-21       ≥ 22
  • Chloramphenicol     30 µg        ≤ 25 26-28   ≥29
  • Co-trimoxazole     25 µg         ≤ 10 11-15    ≥ 16

Key Notes

  1. 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.
  2. 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. influenzae and 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.

Satellitism test for Haemophilus

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. Haemophilus is 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-

Further Readings

  1. https://apps.who.int/iris/rest/bitstreams/910485/retrieve
  2. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Testing; Twentieth Informal Supplement M100-S20. Volume 30 Number 1, Clinical and Laboratory Standards Institute, Villanova, PA. 2010.
  3. Centers for Disease Control and Prevention. Manual for the laboratory Detection of Antimicrobial Resistance among Community-acquired Bacterial Pathogens of Public Health Concern in the Developing World. World Health Organization.WHO/CDS/ CSR/ EPH.2002.15
  4. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  5. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  6. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  7. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  8. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  9. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  10.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
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