Objectives:

The use of prosthesic materials for temporary or permanent implantation has been accompanied by the emergence of implant-associated infections, which are difficult to eradicate. Standard in vitro susceptibility tests only evaluate antimicrobial agent activity against planktonic bacteria and do not evaluate adherent bacteria. In the present study we have developed a simple and reproducible method to evaluate in vitro bactericidal activity of several antimicrobial agents against sessile bacteria. Moreover, bactericidal activities of these antimicrobial agents against planktonic and adherent bacteria have been evaluated.

Methods:

Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were used. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by broth microdilution method, according to NCCLS guidelines. The antimicrobial agents tested were: ampicillin (AMP), cefuroxime (CFX), cefotaxime (CTX), ciprofloxacin (CIP), gentamicin (GNT), trimethoprim/sulfamethoxazole (SXT), imipenem (IMP) and meropenem (MRP) against E. coli, and piperacillin (PIP), CIP, GNT, tobramycin (TBM), ceftazidime (CTZ), cefepime (FEP), IMP and MRP, against P. aeruginosa. MBC of adherent bacteria (MBCADH) was determined from the 96-well plates used for MIC and MBC determinations. Plates were washed two times with cold PBS. At this time, 150 mL of PBS were added to each well, the plates were sealed and adherent bacteria were detached by sonication. Surviving bacteria were determined by plating. All experiments were performed five times.

Results:

With E. coli the MBCADH values of AMP, CFX and CTX were 32, 512 and 64 times higher than MBC of suspended bacteria, respectively. Carbapenems-MBCADH values were 512 times higher than MBC. MBCADH values of CIP, GNT and SXT were 32, 64 and 256 times higher than MBC, respectively. With P. aeruginosa, MBCADH values of PIP, CTZ, FEP and both carbapenems were 256 times higher than MBC. CIP-MBCADH values were also 256 times higher than MBC and aminoglycosides-MBCADH values were 512 times higher than MBC.

Conclusion:

The developed method is simple and reproducible to evaluate in vitro activities of tested antimicrobial agents against sessile bacteria. For all antimicrobial agents tested, and with both strains, the MBC of adherent bacteria was always much higher than MBC of suspended bacteria.