Inhibition of biofilm formation by esomeprazole in Pseudomonas aeruginosa and Staphylococcus aureus

Staphylococcus aureus and Pseudomonas aeruginosa are common nosocomial pathogens responsible for biofilm-associated infections. Proton pump inhibitors (PPI), such as esomeprazole, may have novel antimicrobial properties. The objective of this study was to assess whether esomeprazole prevents sessile bacterial growth and biofilm formation and whether it may have synergistic killing effects with standard antibiotics. The antibiofilm activity of esomeprazole at 0.25 mM was tested against two strains each of S. aureus and P. aeruginosa. Bacterial biofilms were prepared using a commercially available 96-peg-plate Calgary biofilm device. Sessile bacterial CFU counts and biomass were assessed during 72 hours of esomeprazole exposure. The killing activities after an additional 24 hours of vancomycin (against S. aureus) and meropenem (against P. aeruginosa) treatment with or without preexposure to esomeprazole were also assessed by CFU and biomass analyses. P. aeruginosa and S. aureus strains exposed to esomeprazole displayed decreased sessile bacterial growth and biomass (P < 0.001, each parameter). After 72 h of exposure, there was a 1-log(10) decrease in the CFU/ml of esomeprazole-exposed P. aeruginosa and S. aureus strains compared to controls (P < 0.001). After 72 h of exposure, measured absorbance was 100% greater in P. aeruginosa control strains than in esomeprazole-exposed strains (P < 0.001). Increased killing and decreased biomass were observed for esomeprazole-treated bacteria compared to untreated controls exposed to conventional antibiotics (P < 0.001, each parameter). Reduced biofilm growth after 24 h was visibly apparent by light micrographs for P. aeruginosa and S. aureus isolates exposed to esomeprazole compared to untreated controls. In conclusion, esomeprazole demonstrated an antibiofilm effect against biofilm-producing S. aureus and P. aeruginosa.     

Effects of mupirocin at subinhibitory concentrations on biofilm formation in Pseudomonas aeruginosa

BACKGROUND: Subinhibitory concentrations of mupirocin can suppress flagellar formation in Pseudomonas aeruginosa. In this study, we evaluated the effect of mupirocin on biofilm formation in P. aeruginosa. METHODS: P. aeruginosa PAO-1 (MIC, >1,024 microg/ml for mupirocin) was used. Viable bacteria adhering or forming biofilm on a Cell Desk were counted and observed by scanning electron microscopy. RESULTS: With or without continuous exposure to 256 microg/ml of mupirocin, counts of adherent and/or biofilm-forming bacteria showed no difference until day 10. Whereas biofilm formation with glycocalyx was observed on day 6-10 without mupirocin, mupirocin reduced biofilm formation and glycocalyx production for 10 days. CONCLUSIONS: Subinhibitory concentrations of mupirocin can reduce biofilm formation in vitro in P. aeruginosa.     

Effects of antibiotics on adherence of Pseudomonas aeruginosa and Pseudomonas fluorescens to human fibronectin

The effect of subminimal inhibitory concentration (1/2 MIC) of antimicrobial agents on the adherence of fluorescent Pseudomonas to human fibronectin (FN) was investigated by examining two Pseudomonas fluorescens and four Pseudomonas aeruginosa strains. The Escherichia coli (MC1061 and JM109) strains, used as negative controls, adhered poorly to FN immobilized on microtiter wells (adherence indices of 0.04 +/- 0.02 and 0.03 +/- 0.01 10(5) CFU/well, respectively). Two P. aeruginosa strains (NK125502 and ER82483) were highly adherent to FN (adherence indices of 3.22 +/- 0.75 10(5) and 3.08 +/- 1.42 10(5) CFU/well, respectively), the two others (ER98513 and ER92581) adhered less efficiently (adherence indices of 0.91 +/- 0.05 10(5) and 0.45 +/- 0.23 10(5) CFU/well, respectively). The two P. fluorescens strains (MF0 and ER74508) were highly adherent to fibronectin (adherence indices of 7.06 +/- 1.00 10(5) and 2.08 +/- 0.67 10(5) CFU/well, respectively). Cefsulodin was the only antimicrobial agent tested that decreased the FN adherence of the majority of the strains: decrease in adherence highly significant (p < 0.01) for the P. fluorescens MF0 strain, significant (p < 0.05) for the P. aeruginosa NK125502, ER98513 and the P. fluorescens ER74508 strains, and not significant for the P. aeruginosa ER92581 strain. The effects of chloramphenicol and polymyxin B on the adherence of Pseudomonas were strain dependent. Gentamicin had no statistically significant effect on bacterial adherence. We conclude that, in addition to its antibacterial activity, cefsulodin could be effective in preventing the adherence of fluorescent Pseudomonas to FN, an important property in the presence of injured epithelium and coated biomaterials.     

In vitro biofilm characterization and activity of antifungal agents alone and in combination against sessile and planktonic clinical Candida albicans isolates

Thirty clinical isolates of Candida albicans were collected from blood or other sterile site infections. Biofilm dry weight and metabolic activity were measured for each isolate. Planktonic and sessile antifungal susceptibilities of each isolate were determined for amphotericin B deoxycholate, caspofungin, and voriconazole. Sessile susceptibilities were determined for the combination of caspofungin/voriconazole. No significant differences in biofilm dry weight or metabolic activity were found between bloodstream and other invasive isolates. Planktonic MIC90 values and sessile MIC90 (SMIC90) values were 0.25 and 2, 0.06 and >256, and 0.5 and 2 microg/mL for amphotericin, voriconazole, and caspofungin, respectively. The SMIC90 of the combination of caspofungin/voriconazole against sessile isolates was 0.5/2 microg/mL. Therefore, the source of invasive C. albicans clinical isolates did not affect in vitro biofilm formation. Susceptibility to antifungal agents decreased when C. albicans was associated with biofilm, and the combination of caspofungin/voriconazole did not appear to provide enhanced activity compared with caspofungin alone.     

Bactericidal activity of cefepime and ceftriaxone tested against Streptococcus pneumoniae

The bactericidal activities of cefepime and ceftriaxone were assessed by testing a contemporary collection of 50 Streptococcus pneumoniae strains. Minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) of cefepime and ceftriaxone were determined, and time-kill studies were performed on 14 selected strains (10 penicillin-resistant, 2-intermediate, and 2-susceptible). Cefepime and ceftriaxone showed essentially identical potency (MIC50, 1 microg/mL and MIC90, 2 microg/mL, for both compounds) and MBC values (MBC50, 1 microg/mL for both). MBC/MIC ratios were < or = 4 for cefepime and < or = 8 for ceftriaxone on 48 (96.0%) strains, and 2 strains (4.0%) displayed MBC/MIC ratios > or = 32 (tolerance) to the 2 cephalosporins. Time-kill curves corroborated the MBC/MIC studies. Cefepime and ceftriaxone bactericidal activity (> or = 3 log10 CFU/mL reduction in inoculum) was demonstrable after 24 h of exposure to 8x MIC for 13 (92.9%) of 14 strains, whereas 1 strain showed approximately 2 log10 CFU/mL reduction. In conclusion, our results indicate that cefepime and ceftriaxone exhibit comparable potency and bactericidal activities when tested against contemporary pneumococcal strains with varying penicillin susceptibility patterns. Both parenteral cephems offer alternative therapeutic choices for the treatment of invasive pneumococcal infections.     

In vitro evaluation of the activity of two doses of Levofloxacin alone and in combination with other agents against Pseudomonas aeruginosa

P. aeruginosa is one of the most difficult to treat pathogens that generally requires combination therapy to prevent the development of resistance. This study evaluated the in vitro activity of two concentrations of levofloxacin (modeled for the 500 mg and 750 mg daily dose) in combination with ceftazidime, cefepime, piperacillin/tazobactam, imipenem, and tobramycin against P. aeruginosa. MICs and time-kill studies were performed against 12 non-duplicate clinical isolates of P. aeruginosa. The percent susceptible for levofloxacin, ceftazidime, cefepime, piperacillin/tazobactam, imipenem, and tobramycin were 67%, 58%, 58%, 67%, 75%, and 100%, respectively. Tobramycin was the most active single agent, killing and maintaining > or =99.9% killing over a 24 h period against all isolates. Levofloxacin 4 microg/mL(750 mg/day) alone reached 99.9% killing and maintain this killing over the time period more often than levofloxacin 2 microg/mL (500 mg/day). No combination was antagonistic and all combinations with tobramycin were indifferent. Overall, levofloxacin 2 microg/mL plus a beta-lactam was synergistic (65%) more often than levofloxacin 4 microg/mL combinations (46%). This was not unexpected due to the increased activity of levofloxacin 4 microg/mL. However, levofloxacin 4 microg/mL combinations maintained a > or =99.9% killing over the entire 24 h period more often than levofloxacin 2 microg/mL combinations (94% vs 83%). The findings from this work suggest that levofloxacin 750 mg/day in combination with another agent active against P. aeruginosa may prove to be clinically beneficial and superior to combinations using lower doses of levofloxacin. In vivo studies are needed to evaluate the clinical significance of these findings.     

Pharmacodynamics of intermittent- and continuous-infusion cefepime alone and in combination with once-daily tobramycin against Pseudomonas aeruginosa in an in vitro infection model.

Cefepime, a fourth-generation cephalosporin, is currently one of the primary agents used in combination with an aminoglycoside when treating Pseudomonas aeruginosa infections. The bactericidal activity of cefepime administered as intermittent doses (IT) or continuous infusion (CI) both alone and in combination with once-daily tobramycin (ODT) against two clinical strains of P. aeruginosa was compared using an in vitro infection model. Cefepime concentrations simulated human pharmacokinetics after a 1-gram Q12 regimen, or a 1-gram loading dose followed by a 2-gram Q24 CI regimen; the ODT regimen mimicked peak concentrations of >/=10 x MIC. All regimen simulations were run in duplicate over 48 h and a growth control (no antimicrobials added) was run concurrently. Strains tested, PSA5 and PSA10, had MICs of 2 and 8 microg/ml to cefepime, respectively; both MICs to tobramycin were 1.0 microg/ml. CI regimens resulted in concentrations approximately 6x and 2x the MIC for PSA5 and PSA10, respectively. The change in log10 colony-forming units (CFU) per milliliter over time for both P. aeruginosa isolates was compared to initial inocula for all treatment regimens. Initial bolus doses of both IT and CI regimens resulted in a similar decrease in the log10 CFU/ml of both organisms over the first 12 h of the study. After subsequent doses, however, both IT regimens showed greatly diminished bactericidal activity, while both CI regimens were persistently bactericidal without the observation of significant regrowth. As a result, a statistical difference in log10 CFU/ml between both IT and CI regimens with and without ODT was realized at 24, 36 and 48 h for each isolate. Unlike IT dosing, CI cefepime alone or in combination with ODT optimizes bactericidal activity by maximizing the percent of the dosing interval that concentrations remained above the MIC resulting in undiminished bacterial inhibition when compared to IT regimens. These data further suggest that CI is the most efficient method of administration of beta-lactam antibiotics.     

Effect of electrical current on the activities of antimicrobial agents against Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis biofilms

Bacterial biofilms are resistant to conventional antimicrobial agents. Prior in vitro studies have shown that electrical current (EC) enhances the activities of aminoglycosides, quinolones, and oxytetracycline against Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis, Escherichia coli, and Streptococcus gordonii. This phenomenon, known as the bioelectric effect, has been only partially defined. The purpose of this work was to study the in vitro bioelectric effect on the activities of 11 antimicrobial agents representing a variety of different classes against P. aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and S. epidermidis. An eight-channel current generator/controller and eight chambers delivering a continuous flow of fresh medium with or without antimicrobial agents and/or EC to biofilm-coated coupons were used. No significant decreases in the numbers of log(10) CFU/cm(2) were seen after exposure to antimicrobial agents alone, with the exception of a 4.57-log-unit reduction for S. epidermidis and trimethoprim-sulfamethoxazole. We detected a statistically significant bioelectric effect when vancomycin plus 2,000 microamperes EC were used against MRSA biofilms (P = 0.04) and when daptomycin and erythromycin were used in combination with 200 or 2,000 microamperes EC against S. epidermidis biofilms (P = 0.02 and 0.0004, respectively). The results of these experiments indicate that the enhancement of the activity of antimicrobial agents against biofilm organisms by EC is not a generalizable phenomenon across microorganisms and antimicrobial agents.     

Antimicrobial activity of tigecycline tested against nosocomial bacterial pathogens from patients hospitalized in the intensive care unit

The antimicrobial activity of tigecycline and selected antimicrobials was evaluated against bacterial pathogens isolated from patients hospitalized in intensive care units (ICUs) worldwide. A total of 9093 isolates were consecutively collected in >70 medical centers in North America (4157), South America (1830), Europe (3034), and the Asia-Australia (72) areas. The isolates were collected from the bloodstream (68.5%), respiratory tract (13.6%), skin/soft tissue (5.5%), and urinary tract (2.0%) infections in the 2000-2004 period, and susceptibility was tested by reference broth microdilution methods. The most frequently isolated pathogens were Staphylococcus aureus (32.1%), Enterococcus spp. (13.7%), coagulase-negative staphylococci (CoNS; 13.0%), Pseudomonas aeruginosa (8.4%), and Escherichia coli (7.9%). All Gram-positive pathogens (5665) were inhibited at < or =1 microg/mL of tigecycline. Resistance to oxacillin was detected in 43.5% of Staphylococcus aureus and in 85.0% of CoNS, and resistance to vancomycin was observed in 18.6% of enterococci. Tigecycline was very active against Enterobacteriaceae (1876 strains tested) with an MIC90 of < or =1 microg/mL, except for Serratia spp. (2 microg/mL). Extended-spectrum beta-lactamase (ESBL) phenotype was detected in 10% of E. coli and 31% of Klebsiella spp., whereas 28% of Enterobacter spp. were resistant to ceftazidime (AmpC enzyme production). These resistance phenotypes did not adversely affect tigecycline activity. Tigecycline and trimethoprim/sulfamethoxazole were the most active compounds against Stenotrophomonas maltophilia (MIC90, 2 and 1 microg/mL respectively). Tigecycline was also active against Acinetobacter spp. (MIC90, 1 microg/mL), but P. aeruginosa showed decreased susceptibility to tigecycline (MIC90, 16 microg/mL). In summary, isolates from ICU patients worldwide showed high rates of antimicrobial resistance. The most alarming problems detected were vancomycin resistance among enterococci, ESBL-mediated beta-lactam resistance and fluoroquinolone resistance among Enterobacteriaceae, and carbapenem resistance among P. aeruginosa and Acinetobacter spp. Tigecycline exhibited potent in vitro activity against most of clinically important pathogenic bacteria (except P. aeruginosa) isolated from ICU patients and may represent an excellent option for the treatment of infections in this clinical environment.     

Comparative time-kill study of doxycycline, tigecycline, sulbactam, and imipenem against several clones of Acinetobacter baumannii

To assess potential alternative options for the treatment of infections caused by Acinetobacter baumannii, we performed time-kill studies of doxycycline and tigecycline using several isolates recovered from patients residing in 10 different cities in Argentina. Imipenem and sulbactam were also included for comparison purposes. Eleven isolates representing 5 distinctive clones, or isolates with different susceptibility patterns within the same clone, were selected. Tubes containing cation-supplemented Mueller-Hinton broth with and without antibiotics were seeded with a log-phase inoculum of roughly 5 x 10(5) CFU/mL. By using the viable counts determined at 2-, 4-, 6-, 8-, and 24-h intervals after inoculation, a 24-h time-kill curve was constructed for each isolate. No bactericidal activity (defined as a >or=3-log(10) CFU/mL decrease in the viable cell counts with respect to the original inoculum) was observed at any time with sulbactam (4 microg/mL) or tigecycline (1 microg/mL), whereas low bactericidal rate (18% of the isolates) was shown for doxycycline (1 microg/mL) and sulbactam (16 microg/mL) after 24 h of incubation. Doxycycline (4 microg/mL) and tigecycline (8 microg/mL) displayed bactericidal activity at 24 h of incubation against 36% and 54% of the isolates, respectively, including the carbapenem-resistant isolate. Corresponding values for imipenem (1 and 4 microg/mL) against the 10 carbapenem-susceptible isolates were 60% and 90%, respectively. The present study confirms the in vitro efficacy of imipenem against A. baumannii, suggests that doxycycline could be a suitable, cost-effective, alternative option in some instances, and sheds light on the potential role of tigecycline in the treatment of infections with this organism.     

A time-kill evaluation of clarithromycin and azithromycin against two extracellular pathogens and the development of resistance

OBJECTIVE: To determine the activity of clarithromycin, its metabolite (14-hydroxyclarithromycin), and azithromycin against Haemophilus influenzae and Staphylococcus aureus using time-kill methodology and to evaluate the susceptibility of the organisms following exposure to various concentrations of the azalide macrolides. DATA SOURCES AND METHODS: Clinical isolates of H. influenzae and S. aureus were obtained from the Clinical Microbiology Laboratory at University Hospital, San Antonio, Texas. Susceptibility testing was performed according to National Committee for Clinical Laboratory Standards guidelines. 14-Hydroxyclarithromycin was added to clarithromycin solutions used for H. influenzae. Time-kill studies were performed using antimicrobial concentrations of 0.25-8x minimum inhibitory concentration (MIC) and an initial inoculum of approximately 10(5) CFU/mL. Samples were plated onto solid agar at 0, 4, 8, 12, and 24 hours. At 0, 12, and 24 hours, samples were then plated onto solid agar incorporated with antibiotic. After incubating plates at 35 degrees C for 24 hours, colony counts were determined. RESULTS: The MICs of clarithromycin and clarithromycin plus 14-hydroxyclarithromycin for H. influenzae were 4 and 2 microg/mL, respectively. For S. aureus, the MIC of clarithromycin was 0.25 microg/mL, and the MIC of azithromycin for both organisms was 1 microg/mL. H. influenzae developed resistance to both macrolides within 12 hours when exposed to sub-MICs of clarithromycin plus 14-hydroxyclarithromycin. However, when exposed to concentrations less than or equal to the MIC of azithromycin, resistance was not conferred to clarithromycin. S. aureus, on the other hand, became resistant to azithromycin and less susceptible to clarithromycin following exposure to sub-MICs of either macrolide. CONCLUSIONS: Clarithromycin and azithromycin elicited a concentration-independent bacteriostatic effect against H. influenzae and S. aureus at concentrations at least two times the MIC. In addition, concentrations maintained above the MIC prevented changes in the susceptibility of H. influenzae and S. aureus to both macrolides.     

Activity of sodium metabisulfite against planktonic and biofilm Staphylococcus species

Biofilm-forming staphylococci cause a majority of intravascular catheter-related infections. We evaluated the effect of sodium metabisulfite, a preservative commonly added to intravenously administered pharmaceuticals as an antioxidant and previously used as a catheter lock solution, on planktonic and biofilm staphylococci at clinically encountered concentrations. Sodium metabisulfite exhibited bactericidal activity against planktonic Staphylococcus aureus, Staphylococcus lugdunensis, and Staphylococcus epidermidis at concentrations of 512, 512, and 1024 microg/mL, respectively. A concentration of 720 microg/mL inhibited cell growth by all 3 species in a biofilm formation assay. However, established S. aureus and S. lugdunensis biofilms showed less than 1.5 log10 decreases in viable cell counts when treated with 720 microg/mL of sodium metabisulfite for 24 h. These in vitro results suggest that the use of sodium metabisulfite as a catheter lock may inhibit staphylococcal colonization of catheters, thereby preventing catheter-related infection.     

Pharmacodynamic assessment of cefprozil against Streptococcus pneumoniae: implications for breakpoint determinations

Cefprozil, an oral semisynthetic cephalosporin, is commonly utilized in the treatment of respiratory-tract infections in children. While this agent has provided acceptable clinical success over a number of years, this study was undertaken to better define its pharmacodynamic profile against Streptococcus pneumoniae. Nineteen clinical isolates of S. pneumoniae were utilized in the neutropenic murine thigh infection model. To simulate the pharmacokinetic profile of cefprozil in children, the renal function of mice was impaired with uranyl nitrate, and a commercially available cefprozil suspension (6 mg/kg of body weight) was administered orally every 12 h. Mice were infected with 10(6) to 10(7) CFU per thigh, and therapy was initiated 2 h later. At 0 and 24 h postinfection, thighs were harvested to determine bacterial density. Survival was assessed during 96 h of therapy. The magnitude of bacterial kill ranged from 0.5 to 4.4 log(10) CFU per thigh over 24 h, and the extent of microbial eradication was dependent on the MIC. Killing of more than 2.6 log(10) CFU per thigh was observed with MICs of < or =3 microg/ml, while either minimal killing or growth was detected with MICs of > or =4 microg/ml. Mortality in untreated control animals was 100%. Animals infected with strains for which the MICs were < or =2 microg/ml survived the infection, whereas MICs exceeding 2 microg/ml resulted in substantial mortality. These studies demonstrate the effectiveness of cefprozil against isolates of the pneumococcus for which the MICs are < or =2 microg/ml using a drug exposure typically observed in children. These data support a susceptibility breakpoint of < or =2 microg/ml for cefprozil.     

Vancomycin in vitro bactericidal activity and its relationship to efficacy in clearance of methicillin-resistant Staphylococcus aureus bacteremia

We examined the relationship between the time to clearance of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia while patients were receiving vancomycin therapy and the in vitro bactericidal activity of vancomycin. Vancomycin killing assays were performed with 34 MRSA bloodstream isolates (17 accessory gene regulator group II [agr-II] and 17 non-agr-II isolates) from 34 different patients with MRSA bacteremia for whom clinical and microbiological outcomes data were available. Vancomycin doses were prospectively adjusted to achieve peak plasma concentrations of 28 to 32 mug/ml and trough concentrations of 8 to 12 microg/ml. Bactericidal assays were performed over 24 h with approximately 10(7) to 10(8) CFU/ml in broth containing 16 microg/ml vancomycin. The median time to clearance of bacteremia was 6.5 days for patients with MRSA isolates demonstrating > or =2.5 reductions in log(10) CFU/ml at 24 h and >10.5 days for patients with MRSA isolates demonstrating <2.5 log(10) CFU/ml by 24 h (P = 0.025). The median time to clearance was significantly longer with MRSA isolates with vancomycin MICs of 2.0 microg/ml compared to that with MRSA isolates with MICs of < or =1.0 microg/ml (P = 0.019). The bacteremia caused by MRSA isolates with absent or severely reduced delta-hemolysin expression was of a longer duration of bacteremia (10 days and 6.5 days, respectively; P = 0.27) and had a decreased probability of eradication (44% and 78%, respectively; P = 0.086). We conclude that strain-specific microbiological features of MRSA, such as increased vancomycin MICs and decreased killing by vancomycin, appear to be predictive of prolonged MRSA bacteremia while patients are receiving vancomycin therapy. Prolonged bacteremia and decreased delta-hemolysin expression may also be related. Evaluation of these properties may be useful in the consideration of antimicrobial therapies that can be used as alternatives to vancomycin for the treatment of MRSA bacteremia.     

Comparative in vitro bactericidal activity between cefepime and ceftazidime, alone and associated with amikacin, against carbapenem-resistant Pseudomonas aeruginosa strains

Fifteen unique isolates of carbapenem-resistant Pseudomonas aeruginosa were selected for time-kill studies to assess the bactericidal activity of cefepime (CFP) and ceftazidime (CZD) (at 4 and 16 microg/mL), alone and associated with amikacin (AMK) (4 microg/mL). CFP proved more active than CZD (p < 0.05, Student's t test). Bactericidal activity after 24-h incubation was only achieved by the combination of CFP (16 microg/mL) plus AMK. The higher in vitro activity of cefepime over that of ceftazidime against imipenem-resistant P. aeruginosa strains highlights the differences of these drugs beyond Enterobacterspp. and Staphylococcus aureus.     

Influence of erythromycin resistance, inoculum growth phase, and incubation time on assessment of the bactericidal activity of RP 59500 (quinupristin-dalfopristin) against vancomycin-resistant Enterococcus faecium.

RP 59500, a mixture of two semisynthetic streptogramin antibiotics (quinupristin and dalfopristin), is one of a few investigational agents currently in clinical trials with inhibitory activity against multiple-drug-resistant strains of Enterococcus faecium. We evaluated the bactericidal activity of this antimicrobial against 30 recent clinical isolates of vancomycin-resistant E. faecium, including 23 erythromycin-resistant (MIC, >256 microg/ml) and 7 erythromycin-intermediate (MIC, 2 to 4 microg/ml) strains. All isolates were inhibited by RP 59500 at 0.25 to 1.0 microg/ml. The bactericidal activity of RP 59500 was markedly influenced by the erythromycin susceptibility of the strains and by several technical factors, such as inoculum growth phase and time of incubation of counting plates. As determined by time-kill methods, RP 59500 at a concentration of 2 or 8 microg/ml failed to kill erythromycin-resistant organisms under any conditions. Bactericidal activity was observed against all seven erythromycin-intermediate isolates when log-phase inocula were used and the cells were counted after 48 h of incubation (mean reductions in viable bacteria for RP 59500 at concentrations of 2 and 8 microg/ml, 3.45 and 3.50 log10 CFU/ml, respectively), but killing was diminished when the plates were examined at 72 h (mean killing, 3.06 and 2.95 log10, CFU/ml, respectively). No bactericidal activity was observed when stationary-phase cultures were used. On the basis of these data, we expect that bactericidal activity of RP 59500 against the multiple-drug-resistant E. faecium strains currently encountered would be distinctly uncommon.     

Enhancement of antistaphylococcal activities of six antimicrobials against sasG-negative methicillin-susceptible Staphylococcus aureus: an in vitro biofilm model

This study was designed to evaluate antimicrobial activities against methicillin-susceptible Staphylococcus aureus in both sessile and planktonic forms and to detect genes associated with this biofilm phenotype. Minimal biofilm inhibition and eradication concentrations (MBIC and MBEC, respectively) were determined by an in vitro biofilm model, and icaA, atlA, and sasG genes were detected by polymerase chain reaction. Vancomycin and tigecycline presented better biofilm inhibitory activity (MBIC range: 4-8 μg/mL) (P ≤ 0.05) and lower MBEC/MIC ratios (P ≤ 0.001) than other antimicrobials. All isolates harbored icaA and atlA, whereas sasG was present only in strong biofilm formers (P ≤ 0.05). Interestingly, antimicrobial activities against sasG- weak biofilm formers were significantly higher than those against sasG+ strong biofilm formers (P ≤ 0.05), demonstrating that number of cells in a biofilm matrix affected the antimicrobial activity, which was also variable, and might be associated with specific genetic determinants. To our knowledge, this was the first study reporting the presence of sasG in clinical isolates of S. aureus in South America.     

Pharmacokinetics and pharmacodynamics of cefepime administered by intermittent and continuous infusion

OBJECTIVE: This study assessed the pharmacokinetics and pharmacodynamics of cefepime administered by intermittent and continuous infusion against clinical isolates of Pseudomonas aeruginosa, Enterobacter cloacae, and Staphylococcus aureus. BACKGROUND: Because beta-lactam antibiotics exhibit time-dependent bactericidal activity and lack prolonged postantibiotic effects against many bacteria, the goal of therapy is to maintain serum drug concentrations above the minimum inhibitory concentration (MIC) for the relevant pathogen over most of the dosing interval. Continuous infusion is a mode of drug administration that can provide serum drug concentrations continuously above the MIC for most bacterial pathogens. METHODS: Twelve healthy volunteers were enrolled. Each received cefepime 2 g by intermittent bolus q12h and, on another day, was randomly assigned to receive 4 or 3 g administered by continuous infusion over 24 hours. RESULTS: For the intermittent regimen, the mean (+/- SD) pharmacokinetic findings were: maximum serum concentration, 112.9 +/- 21.1 microg/mL; minimum serum concentration, 1.3 +/- 0.5 microg/mL; and half-life, 2.6 +/- 0.4 hours. For the 3- and 4-g continuous infusion regimens, steady-state serum concentrations (C(SS)) were 13.9 +/- 3.8 and 20.3 +/- 3.3 microg/mL, respectively. MICs ranged from 2 to 4, 0.125 to 8, and 2 to 8 microg/mL against P. aeruginosa, E. cloacae, and S. aureus, respectively. For the intermittent regimen, serum inhibitory titers (SITs) at 24 hours were > or = 1:2 in 46% of subjects against P. aeruginosa, 48% against E. cloacae, and 2% against S. aureus. For both continuous infusion regimens, SITs for each organism were > or = 1:2 in all subjects. CONCLUSIONS: The intermittent regimen maintained serum concentrations above the MIC for P. aeruginosa and E. cloacae in > or = 92% (11/12) of subjects for > or = 70% of the dosing interval, provided the MIC was < or = 4 microg/mL. Both continuous infusion regimens provided a C(SS) above the MIC for all organisms. However, the C(SS) was > or = 4 times the MIC only if the MIC was < or = 2 microg/mL. Only the 4-g regimen provided such concentrations against isolates with an MIC of 4 microg/mL, and neither regimen provided such concentrations when the MIC was 8 microg/mL. These findings should be applied in comparative clinical studies.     

Multidrug-resistant Pseudomonas aeruginosa T

The emergence of Pseudomonas aeruginosa strains resistant to carbapenems, fluoroquinolones and amikacin, which were defined as multidrug-resistant (MDR) strains, is a serious problem in Japan. Nosocomial outbreaks of MDR P. aeruginosa infection have become problematic in hospitals. The percentage of MDR isolates at medical facilities was about 2.5% of P. aeruginosa isolates. Furthermore, clonal expansion of P. aeruginosa strains highly resistant to these antibiotics with MICs of more than 64 microg/mL occurred in community hospitals. Most of these multidrug highly resistant strains produced metallo-beta-lactamase IMP-1 and aminoglycoside 6'-N-acetyltransferase AAC(6')-Iae. An immunochromatographic assay was developed for the rapid detection of these enzymes producing strains. The developed assay is an easy-to-use and reliable detection method for the multidrug highly resistant P. aeruginosa.     

Bacteriophage-Mediated Control of a Two-Species Biofilm Formed by Microorganisms Causing Catheter-Associated Urinary Tract Infections in an In Vitro Urinary Catheter Model

Microorganisms from a patient or their environment may colonize indwelling urinary catheters, forming biofilm communities on catheter surfaces and increasing patient morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated silicone catheters with mixtures of Pseudomonas aeruginosa and Proteus mirabilis bacteriophages on the development of single- and two-species biofilms in a multiday continuous-flow in vitro model using artificial urine. Novel phages were purified from sewage, characterized, and screened for their abilities to reduce biofilm development by clinical isolates of their respective hosts. Our screening data showed that artificial urine medium (AUM) is a valid substitute for human urine for the purpose of evaluating uropathogen biofilm control by these bacteriophages. Defined phage cocktails targeting P. aeruginosa and P. mirabilis were designed based on the biofilm inhibition screens. Hydrogel-coated catheters were pretreated with one or both cocktails and challenged with approximately 1 × 10³ CFU/ml of the corresponding pathogen(s). The biofilm growth on the catheter surfaces in AUM was monitored over 72 to 96 h. Phage pretreatment reduced P. aeruginosa biofilm counts by 4 log₁₀ CFU/cm² (P ≤ 0.01) and P. mirabilis biofilm counts by >2 log₁₀ CFU/cm² (P ≤ 0.01) over 48 h. The presence of P. mirabilis was always associated with an increase in lumen pH from 7.5 to 9.5 and with eventual blockage of the reactor lines. The results of this study suggest that pretreatment of a hydrogel urinary catheter with a phage cocktail can significantly reduce mixed-species biofilm formation by clinically relevant bacteria.