Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis

Abstract

Pseudomonas aeruginosa is the predominant cause of chronic airway infection in cystic fibrosis (CF). CF airway isolates are often tested for antibiotic susceptibility but are rarely eradicated by the antibiotics identified as potentially effective. The growth state of P. aeruginosa in CF airways is probably different from that exhibited under conventional susceptibility testing conditions and may represent a bacterial biofilm. Biofilm susceptibility testing methods were adapted to create an assay for implementation in a clinical microbiology laboratory. This assay gave reproducible results when examined in 300 paired determinations with 12 antimicrobial agents, with a serious error rate of 5.7%. The biofilm assay was used retrospectively to test these 12 agents against 94 isolates from 41 CF patients. The biofilm inhibitory concentrations (BICs) were much higher than the corresponding conventionally determined MICs for the beta-lactam antibiotics (median values: aztreonam, >128 microg/ml versus 4 microg/ml; ceftazidime, 128 microg/ml versus 2 microg/ml; piperacillin-tazobactam, 256 microg/ml versus 4 microg/ml; and ticarcillin-clavulanate, 512 microg/ml versus 16 microg/ml, respectively) and doxycycline (>64 microg/ml versus 16 microg/ml); and similar for meropenem (4 micro g/ml versus < or = 1 microg/ml), ciprofloxacin (0.5 microg/ml versus 1 microg/ml), and the aminoglycosides amikacin (32 microg/ml versus 16 microg/ml), gentamicin (16 microg/ml versus 8 microg/ml), and tobramycin (4 microg/ml versus 2 microg/ml). The median BIC for azithromycin was 2 microg/ml, whereas isolates were uniformly resistant when tested by standard methods. This demonstrates the feasibility of adapting biofilm susceptibility methods to the clinical microbiology laboratory and opens the way to examining whether biofilm testing might be used to select more effective antibiotic combinations for CF airway infections than methods in current use.

Figures

FIG. 1.

Biofilm susceptibility assay. See the text for details.

FIG. 2.

Effect of media on biofilm growth. Biofilms of 12 P. aeruginosa CF isolates were grown on pegs in three different media (M63 minimal medium plus 0.4% arginine, gray bars; CAMHB, solid bars; and CAMHB plus 0.4% arginine, open bars) and stained with crystal violet. Bars represent the mean OD590 values, and error bars represent the standard deviations. Isolates 1 to 6 were nonmucoid, and isolates 7 to 12 were mucoid, with eight replicates tested per isolate. The P values from pairwise comparisons included the following (unadjusted for multiple comparisons): CAMHB versus minimal (P = 0.02), CAMHB plus arginine versus minimal (P = 0.04), and CAMHB versus CAMHB plus arginine (P = 0.63).

FIG. 3.

Comparison of sheer stress versus static culture. P. aeruginosa biofilms of 12 isolates were grown under sheer stress (solid bars) and statically (gray bars), and bacteria on pegs were stained with crystal violet. Each value represents the mean OD590 and standard deviation of eight replicates of each isolate (P = 0.07).

FIG. 4.

Comparison of sonication versus centrifugation. Quantification of bacteria removed from the pegs by sonication for 5 min (solid bars) and centrifugation for 20 min at 805 × g (gray bars), with the same set of 12 isolates examined in Fig. 3. Each value represents the mean log10 CFU/peg and standard deviation of eight replicates of each isolate. On average, the density was 0.6 log10 higher with sonication than with centrifugation (P = 0.0001). The 95% confidence interval for the log10 difference in density was 0.4 to 0.8.