Clinical Laboratory Standards Agar Dilution Research Articles

Comparative in vitro activity and the inoculum effect of ertapenem against Enterobacteriaceae resistant to extended-spectrum cephalosporins

The in vitro activity and the inoculum effect of ertapenem were evaluated against a total of 70 Enterobacteriaceae isolates resistant to extended-spectrum cephalosporins. The extended-spectrum β-lactamase phenotypic confirmatory disk diffusion test was performed and AmpC-inducible species were detected using cefoxitin/cefotaxime disk antagonism tests. β-Lactamases were characterised by isoelectric focusing and TEM-specific polymerase chain reaction. Minimum inhibitory concentrations (MICs) were determined by the National Committee for Clinical Laboratory Standards agar dilution method. Ertapenem showed excellent activity against almost all isolates tested, with MIC 50 and MIC 90 values of 0.03 mg/L and 0.12 mg/L, respectively. When the inoculum was increased 100-fold, susceptibility decreased from 98.6% to 65.7% for cefepime and from 75.7% to 54.3% for piperacillin/tazobactam, without changing for ertapenem. The data from this study suggest that this new carbapenem may be useful for treating mixed infections involving Enterobacteriaceae isolates resistant to third-generation cephalosporins.

Antimicrobial potentiality of the thioxanthene flupenthixol through extensive in vitro and in vivo experiments

The antipsychotic thioxanthene flupenthixol, possessing a trifluoromethyl substituent at position 2, exhibited a distinct antibacterial property against 352 strains of bacteria from 3 Gram-positive and 13 Gram-negative genera. The minimum inhibitory concentration (MIC) of flupenthixol was determined by the National Committee for Clinical Laboratory Standards agar dilution method. MICs ranged from 10–100 μg/mL in most of the strains, whilst some strains were inhibited at even lower concentrations. The mode of action of this drug was found to be bacteriostatic against Staphylococcus aureus and Vibrio cholerae. In the in vivo experiments, this drug was capable of contributing significant protection ( P < 0.001) to a Swiss strain of white mice challenged with 50 median lethal dose of a mouse-virulent strain at a drug concentration of 15 μg/mouse. In addition, flupenthixol remarkably reduced the number of viable bacteria in organ homogenates and blood of mice treated with this drug.

Antimicrobial susceptibility profile of molecular typed cystic fibrosis Stenotrophomonas maltophilia isolates and differences with noncystic fibrosis isolates

Multiresistance in Stenotrophomonas maltophilia limits the effectiveness of antimicrobial therapy for infections due to this organism. It can be of special concern in cystic fibrosis (CF) patients due to frequent antimicrobial administration. The in vitro activity of 41 antimicrobial agents against 76 epidemiologically defined CF S. maltophilia isolates by pulsed-field-gel electrophoresis (PFGE) technique under XbaI and SpeI restriction was compared with that obtained with 51 non-CF strains recovered from respiratory sources. Minimal inhibitory concentrations (MICs) were determined with the standard National Committee for Clinical Laboratory Standards agar dilution technique, but with 24-hr incubation. Forty-seven different PFGE profiles were observed within 76 S. maltophilia CF isolates. Minocycline (resistance rate, 0%; MIC(90), 1 microg/ml), doxycycline (6.4%; 8 microg/ml), trovafloxacin (4.2%; 2 microg/ml), moxifloxacin (6.3%; 2 microg/ml), clinafloxacin (6.3%; 2 g/ml), and moxalactam (17.0%; 64 g/ml) displayed low resistance rates. On the contrary, resistance rates were higher with ceftazidime (70.0%; 256 microg/ml), cefepime (83.0%; 128 microg/ml), piperacillin (87.2%; >1,024 microg/ml), ticarcillin (87.2%; >512 microg/ml), and aztreonam (95.7%; >1,024 microg/ml). Clavulanate reverted resistance to ticarcillin and aztreonam in 40.4% and 31.7% of strains, respectively. Aminoglycosides displayed reduced activities with susceptibility rates lower than 20% and MIC(90) higher than 128 microg/ml. With the exception of trimethoprim-sulfamethoxazole (25.4 vs. 31.3%), CF isolates were more resistant than non-CF isolates. Remarkably, resistance was enhanced in S. maltophilia isolates persistently recovered in chronically colonized patients. Susceptibility analysis demonstrated higher resistance rates among CF S. maltophilia isolates when compared with respiratory isolates from non-CF patients. Moreover, persistently recovered CF S. maltophilia isolates were more resistant than sporadic non-CF isolates.

Oxacillin susceptibility testing of coagulase-negative staphylococci using the disk diffusion method and the Vitek GPS-105 card

One hundred and ninety-three isolates of coagulase-negative staphylococci (CoNS) were tested against oxacillin by agar dilution, disk diffusion, and Vitek (GPS-105 card), and the presence of the mecA gene determined by multiplex PCR. The results obtained by all testing methods were in agreement for 190 isolates. Two mecA-negative isolates ( S. lugdunensis and S. haemolyticus) had MICs of ≤0.25 μg/ml by agar dilution and Vitek but were resistant by disk diffusion. One mecA-positive isolate was resistant by Vitek and disk diffusion but had an agar dilution MIC of ≤0.25 μg/ml. For the species of CoNS tested, oxacillin susceptibility results obtained with the Vitek GPS-105 card and disk diffusion correlated well with results obtained by National Committee for Clinical Laboratory Standards agar dilution and with the presence of the mecA gene.

Evaluation of the Etest for penicillin susceptibility testing of Neisseria meningitidis

Susceptibility to penicillin of 30 strains (one isolate per patient) of Neisseria meningitidis isolated from blood ( N = 19) or cerebrospinal fluid ( N = 11) was studied by two methods. Minimum inhibitory concentrations (MICs) obtained with the Etest were compared to those obtained by the National Committee for Clinical Laboratory Standards agar dilution method. Twenty meningococci (67%) relatively resistant to penicillin were identified by both methods. The mean MIC from the reference method was 0.32 μg/ml (range, 0.2 – 1) and by the Etest method was 0.35 μg/ml (range, 0.19 – 1.25). All MICs obtained by the Etest method were within one dilution of the MICs obtained by the reference method. Because of the increase in penicillin MIC of meningococcal isolates in Spain, we evaluated the performance of the Etest as an alternative method for penicillin-susceptibility testing of N. meningitidis. The Etest is a simple and accurate method for determining the susceptibility of N. meningitidis to penicillin.

Activity of WY-49605 compared with those of amoxicillin, amoxicillin-clavulanate, imipenem, ciprofloxacin, cefaclor, cefpodoxime, cefuroxime, clindamycin, and metronidazole against 384 anaerobic bacteria

The National Committee for Clinical Laboratory Standards agar dilution method was used to compare the in vitro activity of WY-49605 (also called SUN/SY 5555 and ALP-201), a new broad-spectrum oral penem, to those of amoxicillin, amoxicillin-clavulanate, imipenem, ciprofloxacin, cefaclor, cefpodoxime, cefuroxime, clindamycin, and metronidazole against 384 clinically isolated anaerobes. These anaerobic organisms included 90 strains from the Bacteroides fragilis group, 87 Prevotella and Porphyromonas strains, non-B. fragilis group Bacteroides strains, 56 fusobacteria, 55 peptostreptococci, 49 gram-positive non-spore-forming rods, and 47 clostridia. Overall, WY-49605 had an MIC range of 0.015 to 8.0 micrograms/ml, an MIC at which 50% of the isolates are inhibited (MIC50) of 0.25 microgram/ml, and an MIC at which 90% of the isolates are inhibited (MIC90) of 2.0 micrograms/ml. Good activity against all anaerobe groups was observed, except for Clostridium difficile and lactobacilli (MIC50s of 4.0 and 2.0 micrograms/ml, respectively, and MIC90s of 8.0 and 2.0 micrograms/ml, respectively). Imipenem had an MIC50 of 0.03 microgram/ml and an MIC90 of 0.25 microgram/ml. Ciprofloxacin was much less active (MIC50 of 2.0 micrograms/ml and MIC90 of 16.0 micrograms/ml). By comparison, all oral beta-lactams were less active than WY-49605, with susceptibilities as follows: amoxicillin MIC50 of 8.0 micrograms/ml and MIC90 of > 256.0 micrograms/ml), amoxicillin-clavulanate MIC50 of 1.0 microgram/ml and MIC90 of 8.0 micrograms/ml, cefaclor MIC50 of 8.0 micrograms/ml and MIC90 of > 32.0 micrograms/ml, cefpodoxime MIC50 of 4.0 micrograms/ml and MIC90 of > 32.0 micrograms/ml, and cefuroxime MIC50 of 4.0 micrograms/ml and MIC90 of > 32.0 micrograms/ml. Clindamycin was active against all groups except some members of the B. fragilis group, Fusobacterium varium, and some clostridia ( overall MIC50 of 0.5 micrograms/ml and overall MIC90 of 8.0 micrograms/ml). Metronidazole was active (MIC of less than or equal to 4.0 micrograms/ml) against all gram-negative anaerobic rods, but most gram-positive non-spore-forming rods, some peptostreptococci, and some clostridia were less susceptible. To date, WY-49605 is the most active oral beta-lactam against anaerobes: these results suggest clinical evaluation for clinical indications suitable for oral therapy.

Comparison of the E-test and conventional agar dilution methods for susceptibility testing of gram-negative anaerobic rods

The E test was used to determine the susceptibility of 209 anaerobic Gram-negative rods to ampicillin, amoxicillin-clavulanate, ticarcillin, ticarcillin-clavulanate, piperacillin, piperacillin-tazobactam, clindamycin, chloramphenicol, and cefoxitin. Minimum inhibitory (MICs) were read where growth intersected the strips or where the upper indentation intersected the strips for β-lactamase inhibitor-containing strips. Reference MICs were performed by the National Committee for Clinical Laboratory Standards agar dilution method. Organisms tested consisted of 117 Bacteroides fragilis group, 24 fusobacteria, and 68 other species (mainly Prevotella species). Agreement of MICs by both methods occurred in 33.4% of cases; 80.8% were within one, 99.0% were within two, and 99.95% were within three doubling dilutions apart. E-test results, expressed as MIC 50/MIC 90 values (μg/ml), were as follows: ampicillin: 16/>256; amoxicillin-clavulanate (2:1), 0.5/4; ticarcillin, 16/>256; ticarcillin-clavulanate (2 μg/ml), 0.5/4; piperacillin, 16/>256; piperacillin-tazobactam (4 μg/ ml), 1 8 ; piperacillinn-tazobactam (8:1), 2 8 ; cefoxitin, 8 64 ; clindamycin, 0.25/4; and chloramphenicol, 2 8 . Conventional MIC methodology yielded MIC 50s and MIC 90s either identical to, or within one doubling dilution of, E-test results. Percentages of strains susceptible varied by s[3%] between the two methods, except for cefoxitin (E test 5.2% lower). Based on these results, the E test is an accurate and practical method for use with the agents and organisms evaluated.

Quality control criteria for testing the susceptibility of anaerobic bacteria to meropenem

Reference values for quality control of in vitro susceptibility tests with meropenem against anaerobic bacteria were determined in a multilaboratory study by the approved National Committee for Clinical Laboratory Standards agar dilution method for the four quality control strains. The study protocol also included the evaluation of microdilution testing, medium additives, and multiple lots of media. The recommended MIC control ranges for three of the control organisms are as follows: Bacteroides fragilis ATCC 25285, 0.06 to 0.125 micrograms/ml; Bacteroides thetaiotaomicron ATCC 29741, 0.125 to 0.5 micrograms/ml; and Eubacterium lentum ATCC 43055, 0.125 to 0.5 micrograms/ml. The modal MIC for Clostridium perfringens ATCC 13124 was at or below the lowest concentration of meropenem tested, and no values are recommended.

Broth-disk elution tests to predict the susceptibility of anaerobic bacteria to the ampicillin-sulbactam combination

The ampicillin-sulbactam Wilkins-Chalgren agar dilution MICs (2 ratios) were compared with the results of broth-disk elution (BDE) tests using various numbers of disks corresponding to elution breakpoints of less than or equal to 8:8 micrograms/ml, less than or equal to 16:8 micrograms/ml, and less than or equal to 16:16 micrograms/ml. This study showed that a 2:1 ratio MIC test and a 16:8 micrograms/ml BDE breakpoint were best for anaerobic organisms; a recommendation consistent with susceptibility testing criteria for rapidly growing aerobic species. The addition of four ampicillin disks (10 micrograms) and four ampicillin-sulbactam discs (10:10 micrograms) to 5 ml of thioglycolate broth was recommended. This test would achieve greater than 99% comparative accuracy to MICs determined with the reference National Committee for Clinical Laboratory Standards agar dilution method.

Comparison of the agar dilution, tube dilution, and broth microdilution susceptibility tests for determination of teicoplanin MICs

The purpose of this study was to evaluate the National Committee for Clinical Laboratory Standards agar dilution, tube dilution, and broth microdilution susceptibility tests for the measurement of teicoplanin MICs. The three standardized tests gave equivalent (within a twofold dilution) results with 98.8 to 99.0% of the 508 gram-positive clinical isolates tested, indicating that either method may be used for teicoplanin MIC determination.

National Committee for Clinical Laboratory Standards agar dilution susceptibility testing of anaerobic gram-negative bacteria.

One hundred nine recent clinical isolates of anaerobic gram-negative bacteria were tested in triplicate by the National Committee for Clinical Laboratory Standards agar dilution procedure for their susceptibility to 32 antimicrobial agents. All isolates were inhibited by imipenem, but there were significant numbers of strains resistant to other beta-lactam drugs, and therefore the in vitro response to these antimicrobial agents cannot be predicted. This was particularly true for the bile-resistant or Bacteroides fragilis group. beta-Lactamase production was detected in 82% of the bacteroides with the nitrocefin test. Clavulanic acid combined with amoxicillin and ticarcillin and sulbactam combined with ampicillin resulted in synergistic activity against all beta-lactamase-positive organisms. Ceftizoxime was the most active of the cephalosporins. Two percent of the isolates were resistant to chloramphenicol and metronidazole. Clindamycin resistance was detected in 38% of the B. fragilis group, which is a marked increase from the 4% detected 10 years ago at this institution.

Rapid recognition of methicillin-resistant Staphylococcus aureus by use of automated test systems.

The ability to rapidly recognize methicillin-resistant Staphylococcus aureus by use of two automated instrument systems, the MS-2 system (Abbott Laboratories, Diagnostics Division, Irving, Tex.) and the AutoMicrobic system (Vitek Systems, Hazelwood, Mo.), was evaluated on a collection of 95 methicillin-resistant S. aureus isolates recovered from at least six geographical areas of the United States. Isolates were simultaneously tested with both systems, and the results were compared with MIC tests performed by the National Committee for Clinical Laboratory Standards agar dilution method. Methicillin-resistant S. aureus isolates were defined as those with a methicillin MIC greater than or equal to 8 micrograms/ml by the reference procedure. Overall, with the AutoMicrobic system, 94.7% of 95 methicillin-resistant S. aureus isolates were detected, and with the MS-2 system, 91.6% of the isolates were detected. Isolates with methicillin MICs greater than or equal to 32 micrograms/ml were readily detected with both systems (41 of 42 isolates). Of 53 isolates from three locales with methicillin MICs of 8 or 16 micrograms/ml, 90.6% (48) were detected by the AutoMicrobic system, whereas 86.8% (46) were detected by the MS-2 system. A program update which has been added to the MS-2 system prints a warning message indicating possible methicillin-resistant S. aureus with isolates which demonstrate multiple antibiotic resistance (greater than or equal to four drugs other than methicillin). This warning message would have provided presumptive recognition of six of eight isolates with discrepant results for methicillin by the MS-2 system.