Measurement of MIC-b, MBIC, and MBEC

For the four frontline antibiotics, levofloxacin, ceftazidime, gentamicin, and trimethoprim, minimum inhibitory concentrations of biofilm (MIC-b), minimum biofilm inhibition concentration (MBIC),and minimum biofilm eradication concentration (MBEC) were determined as described elsewhere.25,26 A total of 21 strong biofilm producing strains simultaneously sensitive to all four antibiotics were selected for biofilm assays. Because of the higher frequency of resistance against trimethoprim only seven strong biofilm former strains simultaneously sensitive to trimethoprim and other three classes of frontline antibiotics could be available (included in total 21 strains). These selected sensitive strains were used for the measurements of MIC-b, MBIC, and MBEC.25 For the measurement of MIC-b, 75 µL of standardized bacterial suspension was inoculated in a 96-well microtiter plate. The plate was incubated at 37°C for 24 hours. After incubation plates were washed with phosphate buffer saline (PBS). Two-fold serial dilutions of all four antibiotics (levofloxacin, ceftazidime, gentamicin, and trimethoprim), ranging from 0.5–2048 µg/mL were prepared and 100 µL of appropriate concentration of each antibiotic was added to each well of a micro-titer plate. The plate was incubated for 18–24 hours at 37°C. MIC-b for each tested sample was visually estimated as the lowest concentration of antibiotic capable of inhibiting biofilm formation of planktonic bacteria. Subsequently, each non-turbid well used to estimate MIC-b was culture plated to assess cell viability.

Likewise, MBIC was determined by inoculating a 75 µL standardized bacterial suspension in a 96 well microtiter plate that was incubated for 24 hours at 37°C. Following the incubation step, medium was removed gently; wells were washed thrice with PBS. A two-fold serial dilution of all four antibiotics (levofloxacin, ceftazidime, gentamicin, and trimethoprim) ranging from 0.5–2048 µg/mL was prepared and a 100 µL of appropriate concentration of each antibiotic was added to each well. Wells were washed and 100 μL of recovery media (MH broth) was added to the wells and incubated for 24 hours at 37°C. The lowest concentration of antibiotic that inhibited re-growth of bacteria was considered MBIC.26 For measurement of MBECs, the treatment procedure was essentially the same as described for MBIC, except after the incubation step, wells without visible growth were scraped thoroughly and particular attention was given to the edges of wells. Scraped material was transferred to 1 mL PBS. Each sample was briefly vortexed to disrupt biofilm and a 100 μL sample was subsequently plated on a fresh tryptic soy agar (TSA) plate. Antibiotic concentration on which no bacterial growth was observed on the TSA plate was considered MBEC.26