Simple fluorometric-based assay of antibiotic effectiveness for Acinetobacter baumannii biofilms

Abstract

Despite strengthened antimicrobial therapy, biofilm infections of Acinetobacter baumannii are associated with poor prognosis and limited therapeutic options. Assessing antibiotics on planktonic bacteria can result in failure against biofilm infections. Currently, antibiotics to treat biofilm infections are administered empirically, usually without considering the susceptibility of the biofilm objectively before beginning treatment. For effective therapy to resolve biofilm infections it is essential to assess the efficacy of commonly used antibiotics against biofilms. Here, we offer a robust and simple assay to assess the efficacy of antibiotics against biofilms. In the present work, we carefully optimized the incubation time, detection range, and fluorescence reading mode for resazurin-based viability staining of biofilms in 96-well-plates and determined minimal biofilm eradication concentrations (MBECs) for A. baumannii isolates from patients with chronic infection. By applying this assay, we demonstrated that antibiotic response patterns varied uniquely within the biofilm formation of various clinical samples. MBEC-50 and 75 have significant discriminatory power over minimum inhibitory concentrations for planktonic suspensions to differentiate the overall efficiency of an antibiotic to eradicate a biofilm. The present assay is an ideal platform on which to assess the efficacy of antibiotics against biofilms in vitro to pave the way for more effective therapy.

Figure 1

(a) Relationship between PrestoBlue reduction (in relative fluorescence units, RFU) and bacterial concentration (in colony forming units or CFU/mL) measured for planktonic bacteria and biofilms. (b) Robustness of the incubation time with PrestoBlue on the antimicrobial susceptibility assay performance, as measured by signal window coefficient, Z′-factor; signal-to-noise (S/N), and signal-to-background (S/B) ratios.

Figure 2

(a) Antibiotic susceptibility of clinical isolates of A. baumannii to seven antibiotics. (b) Distribution of the resistance among various biofilm production capacities as a percentage.

Figure 3

Relationship between susceptibility of A. baumannii clinical isolates and ten antibiotics (1, gentamicin; 2, amikacin; 3, ciprofloxacin; 4, ceftriaxone; 5, colistin; 6, fosfomycin; 7, ceftazidime; 8, imipenem; 9, meropenem; 10, sulbactam).

Figure 4

Association between the level of biofilm formation (negative, weak, moderate, or strong) and susceptibility of A. baumannii clinical isolates test results for ten antibiotics. (a) gentamicin, (b) amikacin, (c) ciprofloxacin, (d) ceftriaxone, (e) colistin, (f) fosfomycin, (g) ceftazidime, (h) imipenem, (i) meropenem, and (j) sulbactam.

Figure 5

Association between the type of A. baumannii clinical isolate sample (1, urine; 2, nasal swabs; 3, tissue; 4, broncho–alveolar aspirates; 5, wound pus; 6, endotracheal aspirates; and 7, sputum) and susceptibility to 10 antibiotics. (a) gentamicin, (b) amikacin, (c) ciprofloxacin, (d) ceftriaxone, (e) colistin, (f) fosfomycin, (g) ceftazidime, (h) imipenem, (i) meropenem, and (j) sulbactam.

Figure 6

Relationship between susceptibility test results, biofilm formation (negative, weak, moderate, or strong) and type of clinical sample (1, urine; 2, nasal swab; 3, tissue; 4-broncho–alveolar aspirate; 5, wound pus; 6, endotracheal aspirates; and 7, sputum).