Cation-supplemented Mueller-Hinton Broth Research Articles

Plasticity of Coagulase-Negative Staphylococcal Membrane Fatty Acid Composition and Implications for Responses to Antimicrobial Agents.

Staphylococcus aureus demonstrates considerable membrane lipid plasticity in response to different growth environments, which is of potential relevance to response and resistance to various antimicrobial agents. This information is not available for various species of coagulase-negative staphylococci, which are common skin inhabitants, can be significant human pathogens, and are resistant to multiple antibiotics. We determined the total fatty acid compositions of Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, and Staphylococcus aureus for comparison purposes. Different proportions of branched-chain and straight-chain fatty acids were observed amongst the different species. However, growth in cation-supplemented Mueller–Hinton broth significantly increased the proportion of branched-chain fatty acids, and membrane fluidities as measured by fluorescence anisotropy. Cation-supplemented Mueller–Hinton broth is used for routine determination of antimicrobial susceptibilities. Growth in serum led to significant increases in straight-chain unsaturated fatty acids in the total fatty acid profiles, and decreases in branched-chain fatty acids. This indicates preformed fatty acids can replace biosynthesized fatty acids in the glycerolipids of coagulase-negative staphylococci, and indicates that bacterial fatty acid biosynthesis system II may not be a good target for antimicrobial agents in these organisms. Even though the different species are expected to be exposed to skin antimicrobial fatty acids, they were susceptible to the major skin antimicrobial fatty acid sapienic acid (C16:1Δ6). Certain species were not susceptible to linoleic acid (C18:2Δ9,12), but no obvious relationship to fatty acid composition could be discerned.

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 × 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 ≥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 μg/mL) or tigecycline (1 μg/mL), whereas low bactericidal rate (18% of the isolates) was shown for doxycycline (1 μg/mL) and sulbactam (16 μg/mL) after 24 h of incubation. Doxycycline (4 μg/mL) and tigecycline (8 μg/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 μg/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.

Susceptibility of beta-hemolytic streptococci to nine antimicrobial agents among four medical centers in Salt Lake City, Utah, USA

A multicenter study was performed to evaluate the susceptibility of beta-hemolytic streptococci to nine antimicrobial agents. MICs were performed in cation-supplemented Mueller-Hinton broth with 3.5% lysed sheep red blood cells according to NCCLS guidelines. A total of 646 isolates were tested: 300 (46%) group A; 170 (26%) group B; 38 (6%) group C, 35 (5%) group F; 83 (17%) group G; and 20 (3%) nongroupable. Six percent of the total isolates were resistant to one or more of the antibiotics tested. Approximately 7% of 387 strains from the University of Utah Hospital and Clinics were resistant to erythromycin. Four isolates were resistant to clindamycin. Six strains (3%) from Primary Children's Medical Center (207 tested) were resistant to one or more of the macrolides. Resistance was rare at the LDS Hospital and the Salt Lake Veteran's Affairs Hospital. Overall, resistance among beta-memolytic streptococci in this geographic location does not seem to be a significant problem, except at the tertiary care university hospital.

Monthly changes in the rate of recovery of penicillin-resistant organisms from children.

Monthly changes in the rate of recovery of penicillin-resistant organisms from children.

Characterization of synergy between ofloxacin, ceftazidime, and tobramycin against Pseudomonas aeruginosa.

The purposes of this study were to investigate the potential for synergy between ceftazidime, tobramycin, and ofloxacin against two clinical isolates (PSA 9258, and PSA 9263) of Pseudomonas aeruginosa utilizing time-concentration-kill curves. A pharmacodynamic model was used to simulate one-compartment pharmacokinetics for single-, double-, or triple-drug combinations utilizing two different elimination half-lives (T1/2). Each duplicate experiment was conducted for 24 h in cation-supplemented Mueller-Hinton broth. Synergy, indifference, and antagonism were defined as reductions of > or = 2.0, > or = 0 to < or = 2, or < or = 0 in mean log10 CFU/ml (CFU = colony-forming units) in bacterial counts at any time point during the 24-hour experiment, respectively. Time-concentration-kill curve studies simulating a peak concentration (CP) = 6 micrograms/ml and T1/2 = 5.5 h for ofloxacin combined with ceftazidime (CP = 80 micrograms/ml; T1/2 = 2 h) resulted in 2.18 and 1.81 log CFU/ml mean decreases in bacterial counts for PSA 9258 and 9263, respectively. Simulated ofloxacin pharmacokinetic parameters (CP = 6 micrograms/ml); T1/2 = 5.5 h) combined with tobramycin (CP = 8 micrograms/ml; T1/2 = 2 h) produced 2.26 and 0.6 log CFU/ml mean reductions in bacterial counts for PSA 9258 and 9263, respectively. Time-concentration-kill curve results were inconsistent with checkerboard synergy experiments which indicated antagonism for ofloxacin/tobramycin combinations (fractional inhibitory concentrations = 2.0/2.5) and indifference for ofloxacin/ceftazidime combinations (fractional inhibitory concentrations = 1.0/1.0). In secondary experiments, tobramycin (T1/2 = 2 h) and ceftazidime (T1/2 = 2 h) at concentrations of one fourth and equal to the minimum inhibitory concentration were combined with ofloxacin (CP = 6 micrograms/ml; T1/2 = 5.5 h). When ofloxacin was combined with tobramycin equivalent to the minimum inhibitory concentration, mean reductions in bacterial counts were 3.74 and 5.59 CFU/ml. These results suggest that an enhanced antipseudomonal activity may result by the combination of clinically achievable concentrations of ofloxacin with minimum inhibitory concentration equivalent concentrations of tobramycin.

Twenty-four-hour area under the concentration-time curve/MIC ratio as a generic predictor of fluoroquinolone antimicrobial effect by using three strains of Pseudomonas aeruginosa and an in vitro pharmacodynamic model.

Several investigators have suggested that the 24-h area under the concentration-time curve (AUC)/MIC ratio (AUC/MIC24 or AUIC24) can be used to make comparisons of antimicrobial activity between fluoroquinolone antibiotics. Limited data exist regarding the generic predictive ability of AUC/MIC24 for the antimicrobial effects of fluoroquinolones. The purposes of the present investigation were to determine if the AUC/MIC24 can be used as a generic outcome predictor of fluoroquinolone antibacterial activity and to determine if a similar AUC/MIC24 breakpoint can be established for different fluoroquinolones. Using an in vitro pharmacodynamic model, 29 duplicate concentration time-kill curve experiments simulated AUC/MIC24s ranging from 52 to 508 SIT-1.h (inverse serum inhibitory titer integrated over time) with ciprofloxacin or ofloxacin against three strains of Pseudomonas aeruginosa. Each 24-h experiment was performed in cation-supplemented Mueller-Hinton broth with a starting inoculum of 10(6) CFU/ml. At timed intervals cation-supplemented Mueller-Hinton broth samples were collected for CFU and fluoroquinolone concentration determinations. Transformation of bacterial counts into the cumulative bacterial effect parameter of the 24-h area under the effect curve (AUEC24) was performed for each concentration time-kill curve. Multivariate regression analysis was used to compare pharmacodynamic predictors (AUC/MIC24, 24-h AUC, peak concentration [Cmax] to MIC ratios [Cmax:MIC], etc.) with ln AUEC24. To identify threshold breakpoint AUC/MIC24s, AUEC24s were stratified by the magnitude of AUC/MIC24 into subgroups, which were analyzed for differences in antibacterial effect. The Kruskal-Wallis test and subsequent Tukey's multiple comparison test were used to determine which AUC/MIC subgroups were significantly different. Multiple regression analysis revealed that only AUC/MIC24 (r2 = 0.65) and MIC (r2 = 0.03) were significantly correlated with antibacterial effect. At similar AUC/MIC24s, yet different MICs, Cmaxs, or elimination half-lives, the AUEC24s were similar for both fluoroquinolones. The relationship between AUC/MIC24 and ln AUEC24 was best described by a sigmoidal maximal antimicrobial effect (Emax) model (r2 = 0.72; Emax = 9.1; AUC/MIC50 = 119 SIT-1.h; S = 2.01 [S is an exponent that reflects the degree of sigmoidicity]). Ciprofloxacin-bacteria AUC/MIC24 values of < 100 SIT-1.h were significantly different (P < 0.05) from the AUC/MIC24 values of > 100 SIT-1.h. An ofloxacin AUC/MIC24 of > 100 SIT-1.h and an AUC/MIC24 of < 100 SIT-1.h exhibited a trend toward a significant difference (P > 0.05 but < 0.1). The inverse relationship between drug exposure and MIC increase postexposure was described by a sigmoidal fixed Emax model (AUC/MIC24, r2 = 0.40; AUC/MIC50 = 95 SIT-1.h; S = 1.97; Cmax:MIC, r2 = 0.41; Cmax:MIC50 = 7.3; S = 2.01). These data suggest that AUC/MIC24 may be the most descriptive measurement of fluoroquinolone antimicrobial activity against P. aeruginosa, that ofloxacin and ciprofloxacin have similar AUC/MIC24 threshold breakpoints at approximately 100 SIT-1.h, that the concentration-dependent selection of resistant organisms may parallel the threshold breakpoint of the antimicrobial effect, and that AUC/MIC24 generically describes the antibacterial effects of different fluoroquinolones.

Comparative effect of protein binding on the killing activities of teicoplanin and vancomycin

The effect of protein binding on the activity of teicoplanin against Staphylococcus aureus was evaluated. Bactericidal rates of teicoplanin in cation-supplemented Mueller-Hinton broth (SMHB) and in a 1:1 mixture of pooled human serum and cation-supplemented Mueller-Hinton broth (PHS-SMHB) were compared with those of vancomycin. Eight concentrations of each drug ranging from 15 to 150 micrograms/ml were studied in two series which correspond to the concentrations in serum achieved with low- (6 mg/kg of body weight once daily) and high-dose (30 mg/kg once daily) teicoplanin. Overall, the bactericidal rate of teicoplanin was lower than that of vancomycin. In the presence of serum, the bactericidal rate of teicoplanin in PHS-SMHB was lower than that in SMHB, often resulting in only one log10 drop in CFU over a 24-h period. There was no statistical difference in the bactericidal rates of high- and low-concentration teicoplanin in either medium. Additionally, concentration-dependent killing in SMHB was not evident with either agent. The bactericidal rates of teicoplanin and vancomycin in a 1:1 mixture of serum ultrafiltrate and SMHB at 60 micrograms/ml were also studied. It was noted that the bactericidal rate of neither agent was affected by the presence of serum ultrafiltrate. This finding is consistent with teicoplanin's high degree of protein binding (reported to be greater than 90% in undiluted serum) and further substantiates the hypothesis that only the free drug is active against microorganisms. These data support protein binding as being a factor in teicoplanin activity against S. aureus.

Stability of viable-bacterium counts in liquid media used for preparation of inocula and subsequent impact on antimicrobial susceptibility test results

We evaluated the consequences of prolonging the time between initial bacterial inoculum suspension preparation and susceptibility test inoculation. Extending the current National Committee for Clinical Laboratory Standards-recommended time of 15 min between suspension preparation and test inoculation should allow laboratories more flexibility to optimize efficiency from the standpoint of workflow. We assessed the length of time for which viable-bacterium counts remain stable in three liquid media at room temperature. Fifty isolates were examined in water, saline, and cation-supplemented Mueller-Hinton broth (CSMHB). Disk diffusion and microdilution MIC tests were performed on nine of these. Our results suggest that directly prepared inoculum suspensions of members of the family Enterobacteriaceae, Pseudomonas aeruginosa, staphylococci, and enterococci can be held for up to 6 h in water or saline prior to inoculation of disk diffusion and MIC tests without compromising test accuracy. The same organisms can be held for at least 1 h in CSMHB. Viridans group streptococci can be held for up to 6 h in saline and CSMHB and for up to 3 h in water. Similarly, Haemophilus influenzae and Streptococcus pneumoniae isolates may be held in CSMHB for up to 3 h. Because of an early decrease in viable-bacterium counts in water and saline with some H. influenzae and S. pneumoniae isolates, we recommend that National Committee for Clinical Laboratory Standards recommendations be followed for these species.

Effect of human serum on the bactericidal activity of daptomycin and vancomycin against staphylococcal and enterococcal isolates as determined by time-kill kinetic studies

The bactericidal activity of daptomycin and vancomycin alone in cation-supplemented Mueller-Hinton broth and in human serum against clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis was evaluated by exposing replicating microorganisms to concentrations ranging from 2 to 128 μg/ml for 24 hr. In addition, the possibility of emergence of resistance, the stability of each agent in the respective medium, and the percent of protein binding by human serum for each agent was evaluated. We found that a concentration of ⩽ 8 μg/ml of daptomycin was sufficient to achieve bactericidal activity (⩾99.9% killing of the inoculum) in cation-supplemented Mueller-Hinton broth for all staphylococcal isolates tested; a concentration of ⩽ 16 μg/ml of daptomycin was required for bactericidal activity in cation-supplemented Mueller-Hinton broth for enterococcal isolates. In human serum, comparable bactericidal activity with daptomycin was achieved only with concentrations 8–16 times higher. A similar but less pronounced effect in human serum was seen for vancomycin. Neither daptomycin nor vancomycin was appreciably degraded in human serum over a 24-hr period. It is likely that the clinical efficacy of daptomycin in humans would be enhanced by higher dosing than has been studied to date.

Wide variability in Pseudomonas aeruginosa aminoglycoside results among seven susceptibility testing procedures

Seven commonly used antimicrobial susceptibility testing methods were used to test the susceptibility of 150 isolates of Pseudomonas aeruginosa against gentamicin, tobramycin, amikacin, carbenicillin, and piperacillin. Results were compared with respect to the susceptibility characteristics of the population of isolates as defined by each method. Conventional methods included agar disk diffusion and agar dilution, carried out in accordance with current recommendations of the National Committee for Clinical Laboratory Standards, as well as broth microdilution testing with cation-supplemented Mueller-Hinton broth (CSMHB). Methods in which instrumentation was used for result determination included the Autobac I, Avantage, Sensititre Autoreader (using a breakpoint panel at 18 h of incubation), and Vitek (AMS-240, using the GNS susceptibility card). When necessary for comparison, MIC data were converted to categorical interpretations (susceptible, intermediate, and resistant). With respect to gentamicin, no significant differences were noted among the results of disk diffusion, broth microdilution, Sensititre Auto breakpoint, or Vitek methods which characterized 60 to 67% of isolates as susceptible, 16 to 22% as intermediate, and 13 to 17% as resistant. In contrast, agar dilution, Autobac, and Avantage, although yielding gentamicin results similar to those of one another, were each significantly different in result reporting from the other four methods above for gentamicin results, and they characterized the Pseudomonas population largely as susceptible (88 to 97%), with 0 to 6% intermediate and only 3% to 6% resistant. More isolates were characterized as being resistant to gentamicin in the Avantage test if an assay broth supplemented with increased amounts of calcium was used. Cation impregnation of Autobac disks did not appreciably change Autobac results. The geometric mean MIC of gentamicin was 4 micrograms/ml lower in the agar dilution method than in the CSMHB microdilution method, despite monitoring of the agar for cation content through performance disk diffusion testing with P. aeruginosa ATCC 27853. Tobramycin activity was greater than gentamicin activity, and susceptibility to tobramycin ranged from 89 to 97%, with few statistically significant differences noted among the seven methods studied. Differences in MIC distribution and geometric mean MIC between agar dilution and CSMHB microdilution testing were minimal and suggested less of a cation influence on tobramycin than gentamicin results. Although amikacin was also more active than gentamicin (83 to 99% of isolates were susceptible), differences in the amikacin results among methods tended to reflect the same trends in reporting as seen with gentamicin testing, with the exception that results of Avantage testing were similar to those of disk diffusion, CSMHB microdilution, Sensititre, and Vitek. A difference in geometric mean MIC of 5 micrograms/ml between CSMHB testing and agar dilution testing suggested the influence of divalent cations on amikacin results. Few highly significant differences were noted among methods when isolates were tested against carbenicillin and piperacillin, except that Avantage piperacillin results (66% susceptible) and Autobac piperacillin results (98% susceptible) were noticeably different from the percent piperacillin susceptibility (range, 85 to 92%) measured by the other methods. Method-dependent variability among aminoglycoside susceptibility results, particularly when testing gentamicin, prevents meaningful comparison of Pseudomonas susceptibility trends among hospitals when different methods are used and promotes confusion and frustration among clinical microbiologists and clinicians owing to the uncertainties of clinical meaning of these data.

Hyperoxia and the antimicrobial susceptibility of Escherichia coli and Pseudomonas aeruginosa

We have tested the ability of hyperoxia (98% O2-2% CO2 at 2.8 atmospheres absolute [ca. 284.6 kPa]) to enhance killing of Escherichia coli (serotype O18 or ATCC 25922) by nitrofurantoin, sulfamethoxazole, trimethoprim, gentamicin, and tobramycin. We have also looked for interactions between hyperoxia and the aminoglycosides against Pseudomonas aeruginosa ATCC 27853. Hyperoxia significantly enhanced bacteriostatic activity of nitrofurantoin and trimethoprim as measured by MIC testing. The possibility exists that these effects might be due to the method required to tests MICs under hyperoxic conditions rather than to the effect of hyperoxia itself. In addition, hyperoxia enhanced killing of bacteria by trimethoprim as measured by MBC testing. Hyperoxia decreased numbers of E. coli by 1.3 log10 and P. aeruginosa by 2.7 log10 in cation-supplemented Mueller-Hinton broth medium. The bacteriostatic effects of hyperoxia did not affect MICs of gentamicin or tobramycin. The lack of interaction between hyperoxia and gentamicin or tobramycin was confirmed by determining the number of viable bacteria remaining after 24 h of exposure to hyperoxia by using a pour plate method. We conclude that hyperoxia potentiates the antimicrobial activity of the reduction-oxidation-cycling antibiotic tested (nitrofurantoin) and of one of the antimetabolites tested (trimethoprim). Hyperoxia does not enhance the bactericidal effects of gentamicin and tobramycin, which require oxidative metabolism for transport into bacterial cells.

Salt-supplemented medium for testing methicillin-resistant staphylococci with newer beta-lactams.

The addition of 2% NaCl to cation-supplemented Mueller-Hinton broth (CSMHB) was evaluated for microdilution testing of the susceptibility of staphylococci to five cephalosporins, imipenem, amoxicillin-clavulanate, and ticarcillin-clavulanate. With Staphylococcus aureus, NaCl improved the recognition of methicillin (oxacillin) resistance to cefamandole, imipenem, or ticarcillin-clavulanate. Resistance to amoxicillin-clavulanate was readily determined, irrespective of the presence of added salt. The addition of 2% NaCl to CSMHB did not significantly improve detection of resistance to any of the beta-lactams among coagulase-negative staphylococci. Since the addition of NaCl did not have significant adverse effects on tests with coagulase-negative staphylococci, the routine addition of 2% NaCl to oxacillin or methicillin tests with staphylococci may be justifiable on the basis of convenience or standardization. However, addition of NaCl to susceptibility tests of other beta-lactams does not consistently improve recognition of resistance among staphylococci and thus cannot be recommended for routine use.

Ceftazidime combined with mecillinam: serum bactericidal titres compared with in-vitro synergy against Gram-negative bacilli

In a study of the possible interaction between mecillinam and ceftazidime against Gram-negative bacilli, ten volunteers received on separate days: ceftazidime 20 mg/kg iv in 15 min, mecillinam 10 mg/kg iv in 15 min, or the combination. Blood samples were obtained before and 1 and 6 h after the end of the infusion. Ten strains each of Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, Salmonella spp. and Yersinia spp. and nine strains each of Acinetobacter spp., and Pseudomonas aeruginosa were selected. Most of the strains were resistant to ampicillin and cefazolin. Serum levels of ceftazidime and mecillinam were measured by bioassay. Serum bacteriostatic (SBS) and bactericidal (SBA) titration was done in microtitre plates in cation supplemented Mueller-Hinton broth and 50% human serum. Chequerboard titration was also studied to assess in-vitro synergy between ceftazidime and mecillinam in Mueller-Hinton broth with or without 50% serum. The mean serum concentrations (SD) were for mecillinam: 6.1 (1.7) at 1 h, and less than 0.3 at 6 h and for ceftazidime: 36.3 (5.5) at 1 h and less than 5 at 6 h. Identical concentrations were measured for the combination. By chequerboard titration, no synergy occurred for Acinetobacter spp. and Ps. aeruginosa, whereas it was observed in 37/60 (FIC) and 33/60 (FBC) of the strains of other species in Mueller-Hinton; from the strains showing synergy, 28/37 (FIC) and 30/33 (FBC) showed also synergy in Mueller-Hinton with 50% human serum. In SBS and SBA, on the other hand, the combination of mecillinam with ceftazidime showed an additive effect against most Enterobacteriaceae tested, synergy being shown for only 10-35% of tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Reliability of high-content disks and modified broth dilution tests for detecting staphylococcal resistance to the penicillinase-resistant penicillins.

In vitro susceptibility tests were performed with 271 isolates of Staphylococcus species (204 Staphylococcus aureus), including 110 strains resistant to the penicillinase-resistant penicillins. Disks containing 5 or 10 micrograms of methicillin, 1 or 4 micrograms of oxacillin, and 1 or 4 micrograms of nafcillin were evaluated. After a full 24 h of incubation at 35 degrees C, tests with 1-microgram oxacillin disks provided optimal results. Use of the more potent oxacillin, nafcillin, or methicillin disks only increased the number of false-susceptible test results. For broth microdilution tests, 2% NaCl should be added to cation-supplemented Mueller-Hinton broth, and MICs should be recorded after a full 24 h at 35 degrees C. Microdilution tests with oxacillin in broth with 2% NaCl were more reliable than similar tests with methicillin.

Effect of NaCl supplementation of Mueller-Hinton broth on susceptibility of staphylococci to aminoglycosides.

The addition of NaCl to cation-supplemented Mueller-Hinton broth increased the MICs of gentamicin, amikacin, and netilmicin for coagulase-positive and -negative staphylococci in a concentration-dependent manner. At 2% NaCl, geometric mean MICs were elevated 14.1- to 25.6-fold. Mueller-Hinton broth containing added NaCl should not be used for testing the susceptibility of staphylococci to aminoglycosides.

Screening method for rapid detection of methicillin-resistant (heteroresistant) Staphylococcus aureus.

A broth screening method was developed to detect methicillin-resistant (heteroresistant) strains of Staphylococcus aureus from primary plates. Lyophilized vials containing cation-supplemented Mueller-Hinton broth, 2% NaCl, and 10 micrograms of methicillin and 6 micrograms of oxacillin per ml were hydrated and tested with 129 methicillin-resistant and 35 methicillin-susceptible strains of S. aureus. With the addition of a tetrazolium indicator after 5 h of incubation, 96.9% of resistant strains were detected. No false-positive results occurred.

In vitro antibacterial activities of antibiotics against Pseudomonas aeruginosa in peritoneal dialysis fluid.

Intraperitoneal antibiotics are used to treat Pseudomonas aeruginosa peritonitis, a serious complication of continuous ambulatory peritoneal dialysis. However, P. aeruginosa killing is often inefficient despite low MBCs. Broth dilution MIC/MBC and time kill curves of tobramycin, amikacin, netilmicin, azlocillin, piperacillin, ceftazidime, cefsulodin, and ciprofloxacin were determined in peritoneal dialysis fluid (PDF), buffered PDF, fluid recovered from patients on continuous ambulatory peritoneal dialysis (RPF), and cation-supplemented Mueller-Hinton broth. MBCs of all antibiotics were 8 to 16 times greater in PDF and RPF than in Mueller-Hinton broth or buffered PDF. Use of the time kill curve technique and Mueller-Hinton broth showed that aminoglycosides killed greater than or equal to 99.9% of P. aeruginosa at 1 h, ciprofloxacin killed greater than or equal to 99.9% at 2 h, and beta-lactams killed greater than or equal to 99.9% at 6 h. In contrast, killing was not demonstrated in PDF by any drug at 6 h and by aminoglycosides only at 24 h. Bactericidal activity was optimal in RPF for ciprofloxacin at 1 h and for aminoglycosides at 2 h; bactericidal activity was not demonstrated in RPF with any beta-lactam (no kill by penicillins; less than 99% kill by cephalosporins). Slow bacterial growth, increased protein binding, and glucose concentrations and other inhibitors may interfere with beta-lactam activity in RPF. These considerations and reported clinical failures and toxicity of aminoglycoside therapy warrant further study of quinolones and drug combinations in P. aeruginosa peritonitis.

Susceptibility testing with the sensititer breakpoint broth microdilution system

The antimicrobial susceptibility profiles of a total of 318 aerobic and facultatively anaerobic bacteria (255 gram-negative bacilli and 63 gram-positive cocci) were determined, using a new commercially available breakpoint broth microdilution procedure (Sensititer Breakpoint System (SBS), Gibco Diagnostics, Inc., Madison, WI) that categorizes test results in the form of susceptibility categories: susceptible, intermediate, and resistant. Results obtained with the SBS were compared with those achieved with a standardized disk diffusion procedure. Among a total of 4,414 organism-antimicrobic comparisons, concordance between the results of the SBS and the disk diffusion procedure was observed in 3,888 cases (88.1%). Four hundred twenty-three (9.6%) minor discrepancies, 45 (1.0%) major discrepancies, and 58 (1.3%) very major discrepancies were noted. Arbitration of major and very major discrepancies with a full-range minimum inhibitory concentration (MIC) procedure confirmed the results of the SBS in 53.4% of cases. A single organism—antimicrobial combination, the nonenterococcal streptococci tested against the aminoglycosides, yielded a significant number of very major errors which were arbitrated in favor of the disk diffusion result. These errors were probably due to poor growth of the test organism in the broth medium used for performing the SBS test (i.e., cation-supplemented Mueller-Hinton broth). With this exception, the SBS was found to be at least as accurate as the standardized disk diffusion procedure.

Early detection of oxacillin-resistant staphylococcal strains with hypertonic broth diluent for microdilution panels.

A total of 292 coagulase-positive and 111 coagulase-negative staphylococcal strains were tested in microdilution MIC panels containing 16 to 0.13 microgram of oxacillin per ml diluted in cation-supplemented Mueller-Hinton broth with and without an additional 2% NaCl. All strains were tested using the stationary-phase inoculum procedure with an incubation temperature of 35 degrees C. Test results were recorded after 16 to 20 h of incubation; staphylococcal strains susceptible to oxacillin (less than or equal to 2 micrograms/ml) were reincubated for 20 to 24 h, and endpoints were determined again. Oxacillin resistance was found in 27 (9%) of the 292 coagulase-positive strains and 39 (35%) of the 111 coagulase-negative strains. Of these resistant strains, 5 (19%) of the 27 coagulase-positive strains and 13 (33%) of the 39 coagulase-negative strains were detected 24 h earlier in cation-supplemented Mueller-Hinton broth with 2% NaCl than in cation-supplemented Mueller-Hinton broth without the additional NaCl. However, 9 (33%) of the 27 resistant coagulase-positive strains and 10 (26%) of the 39 resistant coagulase-negative strains were detected only after an additional 24 h of incubation. Oxacillin MICs for the 265 coagulase-positive susceptible strains and 72 coagulase-negative susceptible strains were not affected by the additional 2% NaCl. These results support the utility of adding 2% NaCl to the broth diluent for the early detection of oxacillin-resistant staphylococcal strains and the necessity of extended incubation for those strains which initially appear to be susceptible to oxacillin after only 16 to 20 h of incubation.

Successful use of broth microdilution in susceptibility tests for methicillin-resistant (heteroresistant) staphylococci.

We studied the broth microdilution antimicrobial susceptibility testing procedure to see whether it could be made reliable for determining resistance of staphylococci to methicillin, oxacillin, nafcillin, and cephalothin. With 45 selected strains of Staphylococcus aureus and 12 selected strains of Staphylococcus epidermidis we found that the addition of 2% NaCl to cation-supplemented Mueller-Hinton broth permitted us to discriminate reliably between resistant and susceptible organisms. A screening test in which resistant staphylococci grew on agar containing 4% NaCl and methicillin (10 micrograms/ml), oxacillin (6 micrograms/ml), or nafcillin (6 micrograms/ml) incubated at 35 degrees C for 24 h (additional 24 h if no growth) was also reliable. In vitro cephalothin resistance occurred in heteroresistant S. aureus but usually did not occur in heteroresistant S. epidermidis.