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Characterization of the inoculum effect with Haemophilus influenzae and beta-lactams
T Balko1, J A Karlowsky, L P Palatnick
1Department of Medical Microbiology, Health Sciences Centre, Winnipeg, Manitoba, Canada.
Abstract:
An inoculum effect is defined as a four-fold or greater increase in MIC with an increase in bacterial inocula. Haemophilus influenzae was tested for an inoculum effect with ampicillin, cefuroxime, and amoxicillin/clavulanate using the standard initial inocula (5 x 10(5) CFU/mL) and a higher initial inocula (1 x 10(7) CFU/mL). An inoculum effect was observed with both beta-lactamase (TEM-1, ROB-1) positive and beta-lactamase negative strains of H. influenzae when MICs were determined based on turbidity. MICs based on viable cell counts however, demonstrated that only beta-lactamase positive strains of H. influenzae produced an inoculum effect. These observations suggest that MICs determined based on turbidity, using high initial inocula, are not reliable when examining the inoculum effect in H. influenzae. The magnitude of the inoculum effect with beta-lactamase positive strains was beta-lactam dependent (ampicillin > amoxicillin/clavulanate > cefuroxime). beta-lactam kill-curves confirmed the aforementioned results. Addition of the beta-lactamase inhibitor clavulanate completely reversed the inoculum effect in beta-lactamase (TEM-1 and ROB-1) positive strains of H. influenzae with all beta-lactams tested. Introduction of the beta-lactamase gene TEM-1 on plasmid vector pLS88 into a beta-lactamase negative strain of H. influenzae (Rd) produced an inoculum effect based on viable cell counts. In conclusion, our results suggest that the beta-lactam inoculum effect demonstrated by H. influenzae is the result of beta-lactamase production and is poorly assessed by turbidity.
Insights
The inoculum effect in Haemophilus influenzae is primarily due to beta-lactamase production, not turbidity. Higher bacterial inocula can inaccurately suggest resistance, especially in beta-lactamase negative strains.
Area of Science:
- Microbiology
- Antimicrobial Resistance
- Pharmacology
Background:
- The inoculum effect, a rise in Minimum Inhibitory Concentration (MIC) with increased bacterial load, impacts antimicrobial susceptibility testing.
- Haemophilus influenzae is a significant human pathogen, and understanding its resistance mechanisms is crucial for effective treatment.
Purpose of the Study:
- To investigate the inoculum effect in Haemophilus influenzae using different beta-lactams and assess the reliability of turbidity-based MIC determination.
- To elucidate the role of beta-lactamase production in the observed inoculum effect.
Main Methods:
- Determining MICs of ampicillin, cefuroxime, and amoxicillin/clavulanate against H. influenzae at standard (5 x 10^5 CFU/mL) and high (1 x 10^7 CFU/mL) inocula.
- Comparing MICs derived from turbidity readings versus viable cell counts.
- Conducting beta-lactam kill-curve studies and transforming beta-lactamase genes into susceptible strains.
Main Results:
- Turbidity-based MICs showed an inoculum effect in both beta-lactamase positive and negative strains, but viable cell counts revealed it only in beta-lactamase positive strains.
- The magnitude of the inoculum effect varied with the beta-lactam tested (ampicillin > amoxicillin/clavulanate > cefuroxime).
- Clavulanate addition abolished the inoculum effect in beta-lactamase positive strains, and introducing the TEM-1 gene induced an inoculum effect in a previously negative strain.
Conclusions:
- The beta-lactam inoculum effect in H. influenzae is predominantly driven by beta-lactamase activity.
- Turbidity-based MIC measurements are unreliable for assessing the inoculum effect in H. influenzae, especially at high bacterial concentrations.
- Accurate assessment of antimicrobial susceptibility requires methods that account for beta-lactamase production and avoid misleading turbidity-based results.