"Affect of anaerobiosis on the antibiotic susceptibility of H. influenzae"

Abstract

Background: Haemophilus influenzae is a human-restricted facultative anaerobe which resides mostly in the oropharynx. The majority of isolates recovered from the throat are unencapsulated commensals (NTHi), but depending on host susceptibility they cause bronchitis, otitis media and on occasion bacteremia and meningitis. Because of the variable oxygen availability in the various niche permitting bacterium replication, the organism must thrive in well oxygenated surfaces, such as pharyngeal epithelium to anoxic environments like the bottom of a Biofilm and in airway mucus. Other reports indicate that H. influenzae use aerobic respiration, anaerobic respiration and fermentation to generate ATP. To gain insight in to the activity of several classes of antibiotics against five well-characterized unencapsulated H. influenzae in room air, in 5% CO2 and under strict anaerobiosis. We also tested for the role of oxidative killing by all cidal antibiotics.

Results: In comparison to room air, testing in 5% CO2 had minimal effects on the susceptibility to aminoglycosides, cephalosporins, tetracycline and chloramphenicol: the MIC of rifampin and ciprofloxacin increased eight fold with certain strains in 5% CO2. All antibiotics, except trimethoprim were cidal under both growth conditions. Aminoglycosides remained bactericidal in a strict anaerobic environment, while a reliable MBC was obtained with trimethoprim only under anaerobic conditions. Kinetic analysis of the cidal action of spectinomycin and tetracycline indicated slower killing anaerobically. An oxidative mechanism for aerobic killing could not be demonstrated.

Conclusions: We conclude that β-lactams, cephalosporins, macrolides, tetracycline's, aminoglycosides, chloramphenicol, rifampin and ciprofloxacin are bactericidal against five well-characterizes H. influenzae in an aerobic and anaerobic environment. The activity of trimethoprim was increased in anaerobic conditions.

Figure 1

A. Strain R2866 was incubated in room air with tetracycline at 2 μg/ml (■), or spectinomycin at 20 μg/ml (▲) with the number of CFU determined on aliquots obtained hourly. Strain R2846 was incubated with 3 μg/ml tetracycline (♦) or spectinomycin at 8 μg/ml (×) with the surviving CFU determined on aliquots obtained hourly. B. Strain R2866 was incubated anaerobically with tetracycline at 2 μg/ml (■), or spectinomycin at 20 μg/ml (▲) with the number of CFU determined on aliquots obtained hourly. Strain R2846 was incubated with 3 μg/ml tetracycline (♦) or spectinomycin at 8 μg/ml (×) with the surviving CFU determined on aliquots obtained hourly.

Figure 2

Hydroxyl ions were detected during an aerobic time-kill assay in room air by measuring the fluorescence at 516_nm of. 3′ (p-hydroxyphenyl), fluorescein (HPF, 5 μM)v Positive controls consisted of 1 mM H₂O₂ added to the media with strain R2866 (×) and R2846 (‡). Hydroxyl ions were not detected during incubation of R2866 with 2 μg/ml tetracycline (■) or Kanamycin at 1.5 μg/ml (▲) or with R2846 in media containing tetracycline at 3 μg/ml (•) or Kanamycin at 1.5 μg/ml (▼).

Figure 3

Strain R2866 was incubated in room air with Kanamycin at 1 μg/ml (×) or tetracycline at 2 μg/ml (♦) in sBHI containing 10 μM bis(1,2-dibutylbarbituric acid) trimethine oxonol [DiBAC_4nm(3)] and the fluorescence measured at 530. Strain R2866 gradually lost membrane permeability during the incubation (●).