Accurate and rapid antibiotic susceptibility testing of Haemophilus influenzae by flow cytometry

Abstract number: p1448

O'Donoghue  M., Pong  W.L., Boost  M.V.

Objectives: 

Haemophilus influenzae continues to cause significant levels of morbidity and mortality and is responsible for serious infections such as pneumonia and meningitis. Susceptibility testing was not required for this organism in the past as it was generally considered sensitive to certain antimicrobials. With the increasing development of resistance in recent years, previously employed empirical therapy is no longer adequate. The fastidious nature of this organism limits the options for susceptibility test methods and requires a minimum of 24 hours turn around time. This study aimed to develop a rapid antimicrobial susceptibility test method (AST) using a flow cytometer (FCM) and to compare the results with standard methods.

Methods: 

Effects of a range of concentrations of ampicillin and tetracycline on a H. influenzae control strain (ATCC 49247) were determined by FCM (Coulter EPICS Elite ESP flow cytometer) using the fluorescent probes DiBAC4 (3) and Propidium iodide (PI). A further 30 clinical isolates of H. influenzae obtained from several district hospitals in Hong Kong were then tested by FCM method to determine their susceptibilities to ampicillin and tetracycline and the results were compared to the Standard Broth Dilution method (SBD). Results obtained were categorized as sensitive, intermediate or resistant using a previously published formula.

Results: 

Treatment with antibiotics resulted in a reproducible and easily detectable change in fluorescence. The degree of change correlated well with sensitivity or resistance as determined by standard methods. There was no difference of categorization of 29 strains tested. The remaining strain being categorized as intermediately resistant by FCM and sensitive by SBD i.e. a minor error.

Discussion: 

Reliable results were obtained within 3 hours allowing the test to be completed within one working day, which may have a considerable clinical impact. The reduction of time for issuing of AST results would enable physicians to choose more appropriate, narrower spectrum and less costly antimicrobial therapy at the initiation of therapy. As reliable results were obtained for both static (tetracycline) and cidal (ampicillin) antibiotics suggesting the method can be extended to other antibiotics. As most hospitals already have a FCM for haematology purposes, it would not be necessary to purchase an additional machine.