Biofilm formation is an important virulence factor that allows bacteria to resist host responses and antibacterial agents. The aim of the study was to assess the in vitro activities of several antimicrobials alone or in combination against two Staphylococcus aureus isolates in a novel pharmacokinetic/pharmacodynamic (PK/PD) model of biofilm for 3 days. One methicillin-susceptible S. aureus strain (SH1000) and one methicillin-resistant S. aureus strain (N315) were evaluated in a modified biofilm reactor with polystyrene coupons. Simulated regimens included vancomycin (VAN) plus rifampin (RIF), moxifloxacin (MOX), and high doses (10 mg/kg of body weight/day) of daptomycin (DAP) alone or combined with RIF or clarithromycin (CLA). Against viable planktonic bacteria (PB) and biofilm-embedded bacteria (BB) of SH1000, neither DAP nor MOX alone was bactericidal. In contrast, the combination of DAP or MOX with CLA significantly increased the activity of the two agents against both PB and BB (P < 0.01), and DAP plus CLA reached the limit of detection at 72 h. Against PB of N315, DAP alone briefly achieved bactericidal activity at 24 h, whereas sustained bactericidal activity was observed at 32 h with VAN plus RIF. Overall, only a minimal reduction was observed with both regimens against BB (<2.8 log 10 CFU/ml). Finally, the combination of DAP and RIF was bactericidal against both PB and BB, achieving the limit of detection at 72 h. In conclusion, we developed a novel in vitro PK/PD model to assess the activities of antimicrobials against mature bacterial biofilm. Combinations of DAP or MOX with CLA were the most effective regimens and may represent promising options to treat persistent infections caused by S. aureus biofilms.

Colistin resistance, although uncommon, is increasingly being reported among Gram-negative clinical pathogens, and an understanding of its impact on the activity of antimicrobials is now evolving. We evaluated the potential for synergy of colistin plus trimethoprim, trimethoprim-sulfamethoxazole (1/19 ratio), or vancomycin against 12 isolates of Acinetobacter baumannii (n ‫؍‬ 4), Pseudomonas aeruginosa (n ‫؍‬ 4), and Klebsiella pneumoniae (n ‫؍‬ 4). The strains included six multidrug-resistant clinical isolates, K. pneumoniae ATCC 700603, A. baumannii ATCC 19606, P. aeruginosa ATCC 27853, and their colistin-resistant derivatives (KPm1, ABm1, and PAm1, respectively). Antimicrobial susceptibilities were assessed by broth microdilution and population analysis profiles. The potential for synergy of colistin combinations was evaluated using a checkerboard assay, as well as static time-kill experiments at 0.5؋ and 0.25؋ MIC. The MIC ranges of vancomycin, trimethoprim, and trimethoprim-sulfamethoxazole (1/19) were >128, 4 to >128, and 2/38 to >128/2,432 g/ml, respectively. Colistin resistance demonstrated little impact on vancomycin, trimethoprim, or trimethoprim-sulfamethoxazole MIC values. Isolates with subpopulations heterogeneously resistant to colistin were observed to various degrees in all tested isolates. In time-kill assays, all tested combinations were synergistic against KPm1 at 0.25؋ MIC and 0.5؋ MIC and ABm1 and PAm1 at 0.5؋ MIC. In contrast, none of the tested combinations demonstrated synergy against any colistin-susceptible P. aeruginosa isolates and clinical strains of K. pneumoniae isolates. Only colistin plus trimethoprim or trimethoprim-sulfamethoxazole was synergistic and bactericidal at 0.5؋ MIC against K. pneumoniae ATCC 700603. Colistin resistance seems to promote the in vitro activity of unconventional colistin combinations. Additional experiments are warranted to understand the clinical significance of these observations.

We evaluated the ability of four commercial MIC testing systems (MicroScan, Vitek 2, Phoenix, and Etest) to detect vancomycin MIC values of <1 to >2 in 200 methicillin-resistant Staphylococcus aureus (MRSA) strains compared to the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference methods. Compared to the BMD method, absolute agreement (0 ؎ dilution) was highest for the Phoenix system (66.2%) and the MicroScan turbidity method (61.8%), followed by the Vitek 2 system (54.3%). The Etest produced MIC values 1 to 2 dilutions higher than those produced by the BMD method (36.7% agreement). Of interest, the MicroScan system (prompt method) was more likely to overcall an MIC value of 1 mg/liter (74.1%), whereas the Phoenix (76%) and Vitek 2 (20%) systems had a tendency to undercall an MIC of 2 mg/liter. The ability to correctly identify vancomycin MIC values of 1 and 2 has clinical implications and requires further evaluation.T he overall prevalence of methicillin-resistant Staphylococcus aureus (MRSA) continues to increase, with vancomycin (VAN) remaining the mainstay of therapy for serious infections (1-3). Over the last several years, there have been a number of studies that have demonstrated an association between vancomycin MICs of 1.5 or 2 mg/liter and failure of vancomycin therapy, even though these values lie within the Clinical and Laboratory Standards Institute's (CLSI's) and the FDA's acceptable vancomycin susceptibility range (Յ2 mg/liter). The majority of these reports were derived from patients with MRSA-complicated bacteremia in whom a vancomycin MIC of 1.5 or 2 mg/liter was associated with persistent signs and symptoms of infection, including prolonged days of bacteremia, increased complications, increased lengths of hospital stay, and mortality (4-9). A recent observational study of 532 patients with MRSA and methicillinsusceptible Staphylococcus aureus (MSSA) bacteremia noted a significantly higher 30-day mortality rate associated with patients who had an isolate for which the MIC exceeded 1.5 mg/liter (by Etest methods). Of interest, this association did not seem to be related to vancomycin treatment, since patients who had MSSA bacteremia with an elevated vancomycin MIC and were treated with flucloxacillin had worse clinical outcomes than flucloxacillin-treated patients with MSSA bacteremia that had a lower vancomycin MIC value (P ϭ 0.012) (10). In addition, a recent systematic review and meta-analysis of the literature regarding the relationship of vancomycin susceptibility and patient outcome concluded that vancomycin MIC values of 1.5 or 2 mg/liter were associated with greater treatment failure and mortality rates in patients with MRSA infections (11). Patients who have serious high-inoculum infections (i.e., infective endocarditis, medical device infections, etc.) with MRSA and for which the strain has been identified as heteroresistant vancomycin-intermediate S. aureus (hVISA) are also more likely to experience prolonged days of bacteremia, increased complications, m...

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