Comparison of fixed concentration and fixed ratio options for testing susceptibility of gram-negative bacilli to piperacillin and piperacillin/tazobactam

Piperacillin combined with tazobactam was tested at both a fixed ratio (8:1) and fixed tazobactam concentration (4 µg/ml) against 2,685 consecutively isolated gram-negative bacilli and 56 highly piperacillin-resistant isolates. Tazobactam significantly enhanced the spectrum of piperacillin activity. Overall, at a concentration of 16 g/ml piperacillin alone inhibited 78.8 % of theEnterobacteriaceae isolates compared to inhibition of 92.7 % and 95.5 % by the 8:1 ratio and fixed (4 µg/ml) tazobactam combinations, respectively. In MIC tests the two combination options performed comparably against both routine and highly piperacillin-resistant isolates. Synergistic inhibition was observed for comparable numbers of isolates with the two combination options, the most marked effect being seen in the more highly piperacillin-resistant isolates. Both testing options are supported by the available human pharmacokinetic data; however the 8:1 ratio of piperacillin to tazobactam may be preferable given that the clinical formulation contains the two compounds in an 8:1 ratio and this ratio is maintained in vivo.     

Spiral gradient endpoint method compared to standard agar dilution for susceptibility testing of anaerobic gram-negative bacilli.

More efficient and reproducible alternative methods of performing agar dilution susceptibility testing are desirable, particularly for anaerobic bacteria. Anaerobes generally grow more reliably on solid media than they do in broth microdilution wells. A new method, the revised spiral gradient endpoint (SGE) method, was evaluated against the standard agar dilution (SAD) method by using a wide variety of anaerobic gram-negative bacilli (161 strains) and eight antimicrobial agents. For the SGE method, a spiral plater was used to set up a concentration gradient of an antimicrobial agent within an agar plate across which bacterial strains were inoculated as radial streaks. After incubation, the MIC of the antimicrobial agent was calculated from the radial endpoint location where bacterial growth ceased along the streak. The MICs for 90% of strains tested (in micrograms per milliliter) and the cumulative percentages of susceptible strains at the breakpoints for the SGE and SAD methods, respectively, and for all 161 strains were as follows: for metronidazole, 2 and 100 versus 2 and 100; for imipenem, 1 and 99 versus 0.5 and 98; for ampicillin-sulbactam, 8 and 97 versus 8 and 98; for clindamycin, 4 and 90 versus 4 and 91; for cefoxitin, 32 and 95 versus 32 and 95; for mezlocillin, 256 and 88 versus greater than 128 and 86; for ampicillin, greater than or equal to 256 and 51 versus greater than 64 and 51; and for penicillin (in units per milliliter), greater than or equal to 512 and 71 versus greater than 64 and 65. The excellent agreement of these data and the greater sensitivity reproducibility, and efficiency of the revised SGE method warrant further evaluations. Assuming that these advantages are confirmed, the revised SGE method should be a useful alternative test method when detailed susceptibility data are desired.     

Comparison of three automated systems for antimicrobial susceptibility testing of gram-negative bacilli.

Several instruments for automated or semiautomated susceptibility testing are currently available. Three of these instruments, Autobac (General Diagnostics, Warner-Lambert Co., Morris Plains, N.J.), MS-2 (Abbott Laboratories, Dallas, Tex.), and AutoMicrobic system (AMS) (Vitek, Inc., Hazelwood, Mo.) were compared for antimicrobial susceptibility testing of gram-negative bacilli. A total of 207 isolates representing 29 species of gram-negative bacilli were tested simultaneously by each instrument and by a standardized disk diffusion reference method. Nine antimicrobial agents, including ampicillin, carbenicillin, cephalothin, gentamicin, tobramycin, amikacin, tetracycline, trimethoprim-sulfamethoxazole, and nitrofurantoin were tested. Discrepancies between the results of the automated and reference disk diffusion methods were resolved by agar dilution testing. Overall, 93% of the Autobac and MS-2 results and 83% of the AMS results were in agreement with the results obtained by the reference methods. The results of the Autobac, MS-2, and AMS systems respectively included 3.3, 2.3, and 4.2% major and very major discrepancies. Excessive testing discrepancies were found for certain drugs, including ampicillin, tetracycline, and nitrofurantoin, and for certain organisms, including species of Providencia, Serratia, and Citrobacter. The results of this comparison of three automated systems for antimicrobial susceptibility testing indicate that the Autobac and MS-2 instruments provided highly reliable results. The AMS need further development of its susceptibility testing capability to eliminate an unacceptably high number of minor discrepancies.     

Comparison of two automated instrument systems for rapid susceptibility testing of gram-negative bacilli.

The Vitek AutoMicrobic System with GSC-plus cards and the Abbott MS-2 system were tested in parallel and the results were compared directly with those of a reference microdilution minimal inhibitory concentration (MIC) procedure on a group of 262 clinical isolates of the family Enterobacteriaceae and of Pseudomonas aeruginosa. Results of both systems were compared with the reference MIC for category agreement, and in addition, the Vitek MICs were compared with those obtained by the reference procedure. The Vitek system provided an essential category correlation of 89.4% for enteric bacteria and 97.0% for P. aeruginosa. Vitek MICs agreed within 1 twofold dilutional increment for 86.3% of the enteric bacteria tested and for 96.2% of the P. aeruginosa isolates. The Abbott MS-2 essential categoric agreement was 92.0% for enteric bacteria and 92.4% for P. aeruginosa. If only aminoglycosides or carbenicillin were considered for P. aeruginosa isolates, the essential category agreement was 92.5% for the Vitek and 93.3% for the MS-2. The majority of MS-2 category errors (13 of 19) with P. aeruginosa involved gentamicin results on isolates whose reference MICs were 8 micrograms/ml and whose MS-2 results were susceptible (MIC less than or equal to 4 micrograms/ml). Retesting of the P. aeruginosa isolates in calcium-supplemented MS-2 broth increased the essential agreement for the aminoglycosides to 97.5%.     

Evaluation of four anti-microbic susceptibility testing systems for gram-negative bacilli

A total of 200 clinical isolates were assayed by five anti-microbic susceptibility testing systems. Two frozen minimal inhibitory concentration (MIC) systems (MicroScan and Pasco), an automated MIC system (AMS, Vitek Systems), and the standard disk diffusion were compared with a reference broth dilution method. Organisms tested included 100 resistant clinical stock strains and 100 fresh random clinical isolates. Overall, there were 1,600 anti-microbic-organism combinations analyzed. The Pasco and MicroScan systems had no major discrepancies, the AMS system had seven, and the disk diffusion two. The number of very major discrepancies were as follows: AMS, 11; disk diffusion, 9; MicroScan, 5; Pasco, 2. Of the total 36 major or very major discrepancies in the study, 33% (12 of 36) were with an aminoglycoside and 44% (16 of 36) occurred with a second-generation cephalosporin, of which 10 of 16 were with cefamandole. Overall, there was a greater than 98.8% essential agreement with all systems compared with the reference method.     

Evaluation of the AutoMicrobic system for identification and susceptibility testing of gram-negative bacilli

The AutoMicrobic system (AMS) (Vitek Systems, Inc., Hazelwood, Mo.) was compared with the API-20E system for the identification of gram-negative bacilli by using 380 stock clinical isolates and 377 immediately encountered fresh clinical isolates. For the stock isolates, with Enterobacteriaceae-Plus Biochemical Cards and automated interpretation, 364 (95.8%) were in agreement to the species level. For the fresh clinical isolates, agreement at the genus and species levels was 89.7 and 85.9%, respectively, when Enterobacteriaceae-Plus Cards were interpreted by the AMS. Manual interpretation of Enterobacteriaceae-Plus Biochemical Cards improved species level agreement to 91.0%. Subsequent retesting of all discrepant isolates with the Gram-Negative Identification Card resulted in significant improvement of results, and for the stock and fresh clinical isolates, species level agreement was 98.7 and 97.3%, respectively. AMS susceptibility testing was evaluated by comparing ampicillin and cephalothin MICs determined in parallel by AMS and a reference broth microdilution test for stock isolates, and by comparison of AMS and standardized disk agar diffusion test results for fresh clinical isolates. For the stock isolates, AMS mean integer MICs approximated microdilution mean integer MICs with AMS, providing excellent MIC replicability. For ampicillin and cephalothin, 50 and 46.8%, respectively, of AMS integer MICs were within +/- 1 microgram/ml of the reference values, and 89.3 and 63.1% of AMS integer MICs were within +/- 2 micrograms/ml of the reference values. For the fresh clinical isolates, AMS and reference results were in disagreement for 4.5% of the antimicrobial agents tested, with 2.3% as a combination of "major" and "very major" errors.     

Evaluation of the AutoMicrobic system for susceptibility testing of aminoglycosides and gram-negative bacilli.

The AutoMicrobic system (AMS; Vitek Systems, Inc., Hazelwood, Mo.) was compared with a reference broth microdilution MIC method to determine the accuracy and reproducibility of aminoglycoside susceptibility testing of gram-negative bacilli. Stock clinical isolates (n = 176) which demonstrated resistance to at least one aminoglycoside, extended-spectrum penicillin, or broad-spectrum cephalosporin (or a combination) were selected for this study. Isolates with moderate susceptibility to the aminoglycosides were also included. Of these isolates, 116 were either resistant or moderately susceptible to one or more of amikacin, gentamicin, netilmicin, and tobramycin. When AMS MIC results for 704 antimicrobial agent-organism combinations were compared with parallel microdilution MIC results, exact agreement (AMS MIC = reference MIC) rates were: amikacin, 71.6%; gentamicin, 71.6%; netilmicin, 83.0%; and tobramycin, 69.3%. Agreement rates within +/- 1 log2 dilution were: amikacin, 96.0%; gentamicin, 93.8%; netilmicin, 97.2%; and tobramycin, 96.0%. When National Committee for Clinical Laboratory Standards criteria were used to qualitatively evaluate performance, the overall agreement rates were: amikacin, 100.0%; gentamicin, 99.4%; netilmicin, 98.9%; and tobramycin, 99.4%. There were only four very major discrepancies, which represented 0.6% of the tests performed, and there were no major discrepancies. The percentages of minor discrepancies were: amikacin, 9.6%; gentamicin, 14.2%; netilmicin, 11.9%; and tobramycin, 10.8%. Of the overall average of 11.6% minor discrepancies, 9.7% occurred even though the AMS MIC was within +/- 1 log2 dilution of the reference MIC. The intralaboratory reproducibility ranged from 93.3 to 100% for the four drugs examined. With this challenge group of gram-negative bacilli, the AMS generated aminoglycoside MIC results that were comparable to those obtained by a reference broth microdilution method.     

Evaluation of the autoSCAN-W/A rapid system for identification and susceptibility testing of gram-negative fermentative bacilli.

The autoSCAN-Walk-Away (W/A) system for identification and susceptibility testing was evaluated for 400 gram-negative fermentative bacteria by using the API 20E (366 isolates) and/or tube biochemical tests as the reference identification system and a frozen microdilution MIC tray system for susceptibility testing. The W/A system performed well for identification of this group of organisms representing 14 genera and 30 species, showing a sensitivity of 96% and results available in 2 h. Of the 16 misidentifications, 6 were with Serratia liquefaciens. A total of 63 isolates (17%) required further tests to complete the identification, compared with 106 (29%) of the isolates which required additional tests for the API 20E identification. Approximately half (32) of the additional tests with the W/A system were required in order to separate Citrobacter diversus from C. amalonaticus. For susceptibility determinations, the W/A system demonstrated an overall agreement of 93% (4,102 determinations) with 40 major errors (0.98%). However, of the 906 resistant organism-drug combinations in the study, there were 115 very major errors, for a false-susceptibility rate of 12.7% of the resistance determinations. Among these very major errors, 80% occurred with piperacillin and the cephalosporins. The W/A system completed the MIC determinations in 7 h; however, the difficulty in detecting resistance with some antimicrobial agents limited the advantages of the rapid susceptibility testing.     

Comparison of the Cathra Repliscan II, the AutoMicrobic system Gram-Negative General Susceptibility-Plus Card, and the Micro-Media System Fox Panel for dilution susceptibility testing of gram-negative bacilli.

A comparative evaluation was done to test the accuracy of the Cathra Repliscan II agar dilution system (Diagnostic Equipment, Inc., St. Paul, Minn.), the AutoMicrobic system with Gram-Negative General Susceptibility-Plus Card (Vitek Systems, Inc., Hazelwood, Mo.), and the Micro-Media Fox Panel micro broth dilution system (Micro-Media Systems, Inc., San Jose, Calif.) in determining MICs of 12 antibiotics for 200 gram-negative bacilli. Of the 200 strains tested, 12 isolates did not grow in one of the three systems. The 188 remaining organisms included 158 members of the family Enterobacteriaceae, 20 Pseudomonas spp., 5 Acinetobacter sp., 3 Aeromonas spp., and 2 Vibrio spp. A total of 2,256 organism-antibiotic combinations were analyzed for each system. An MIC was considered correct if two of the three systems were in agreement. When disagreements occurred, correct MICs were determined by the standard agar dilution method. With this criterion, overall agreements of the Cathra Repliscan II system, AutoMicrobic system, and Micro-Media Fox Panel system were 94.7, 94.9, and 95.5%, respectively. Tetracycline (20%), nitrofurantoin (20%), and ampicillin (16%) accounted for 56% of the discrepancies observed. These results indicate that all three systems perform with a high degree of accuracy for susceptibility testing of gram-negative bacilli.     

Rapid antimicrobial susceptibility testing of gram-negative bacilli using Baxter MicroScan rapid fluorogenic panels and autoSCAN-W/A

The MicroScan Rapid Neg MIC/Combo panels and autoSCAN-W/A (Walk Away) system utilize automated fluorescence technology for rapid antimicrobial susceptibility testing of Gram-negative bacilli. In a three site clinical study eleven antimicrobial agents were evaluated by comparing results obtained with 741 clinical isolates, using rapid fluorogenic expanded dilution MIC panels and corresponding frozen microdilution reference panels determined visually. Results for 31%, 40%, 12% and 9% of the isolates were available within 3.5, 4.5, 5.5 and 7.0 hours respectively. Results for 7.3% were not available within that time period. For the seven drugs analyzed using a Minimum Inhibitory Concentration range of dilutions, overall agreement (+/- 1 dilution) was 94%, with 1.5% very major, 0.9% major and 2.5% minor errors. For the four drugs analyzed using a Breakpoint range of dilutions, overall agreement (+/- 1 dilution) was 97%, with two percent very major, and one percent major errors. The MicroScan Rapid Neg MIC system is an accurate and rapid method for same day determination of susceptibility of Gram-negative bacilli.     

Evaluation of the Autoscan Walkaway system for rapid identification and susceptibility testing of gram-negative bacilli.

We evaluated the performance of the Autoscan Walkaway (W/A) system (MicroScan, Sacramento, Calif.) in conjunction with the fluorometric Neg Combo panels for rapid identification and susceptibility testing of gram-negative bacilli. Fermentative and nonfermentative gram-negative bacilli were tested in parallel with the W/A system and the Cathra Repliscan replicator (C/R) system (Cathra, St. Paul, Minn.). Conventional biochemical testing and agar dilution testing were used to resolve the identification and susceptibility testing discrepancies. Of 495 clinical isolates tested, 445 (90%) were correctly identified by the W/A system and 483 (98%) were correctly identified by the C/R system. Repeat testing by using updated versions of the W/A system's computer identification software failed to demonstrate improved identification accuracy. For susceptibility testing, the W/A system demonstrated 5.6% total interpretative category errors, including only 0.9% major and very major errors. The comparative C/R system produced only 1% errors overall, including 0.2% major and very major errors. Although the W/A system is highly automated and is capable of producing results rapidly, our findings suggest that additional identification and susceptibility testing refinements are needed before the system will be suitable for routine use.     

Comparison of ampicillin/sulbactam and amoxicillin/clavulanic acid for detection of borderline oxacillin-resistantStaphylococcus aureus

Borderline oxacillin-resistantStaphylococcus aureus (BORSA) may be misidentified as intrinsically methicillin-resistantStaphylococcus aureus (MRSA) in the clinical laboratory. Under disk diffusion testing conditions designed to maximize detection of MRSA (incubation at 32 °C, pre-induction with methicillin, or plating on 4 % NaCl-supplemented agar), BORSA strains also tend to appear resistant to semisynthetic penicillins. Under these conditions, ampicillin/sulbactam appears to be more accurate than amoxicillin/clavulanic acid for differentiating BORSA from MRSA.     

Response of enteric gram-negative bacteria to disks containing 20 micrograms each of ampicillin and sulbactam.

Ampicillin-sulbactam disks containing either 10 microg of each drug or 20 microg of each drug were tested against 138 recently, sequentially isolated members of the family Enterobacteriaceae. Results obtained with the higher-content disks corresponded more closely to the impressions of clinicians.     

Comparison of Etest, Vitek and agar dilution for susceptibility testing of colistin

In total, 172 isolates of Enterobacteriaceae, Acinetobacter spp., Pseudomonas aeruginosa and Stenotrophomonas maltophilia were tested for susceptibility to colistin by agar dilution, Etest and the Vitek 2 system. Isolates with a colistin MIC < or =2 mg/L were considered to be susceptible. Fifty-four (31%) Gram-negative isolates were resistant to colistin. Categorical agreement between agar dilution and Etest was 87%, and between agar dilution and Vitek 2 was 82%. Based on the data obtained, the Vitek 2 system was unreliable for detecting colistin resistance, and results obtained by Etest may require confirmation by a standard MIC susceptibility testing method.     

Comparison of media for agar dilution susceptibility testing ofBordetella pertussis andBordetella parapertussis

Antimicrobial susceptibility testing of the fastidious speciesBordetella pertussis andBordetella parapertussis is not standardized. In an attempt to find the optimal medium for agar dilution testing, the activity of erythromycin againstBordetella pertussis andBordetella parapertussis (34 isolates each) was assessed using homologous broth/agar combinations of Bordet-Gengou, charcoal, Iso-Sensitest (Oxoid) and Mueller-Hinton media. Each medium was supplemented with 5 % and 20 % whole defibrinated horse blood. Mueller-Hinton medium supplemented with 5 % horse blood performed best overall.     

Direct identification and susceptibility testing by the AutoMicrobic system of gram-negative bacilli from urine specimens.

A total of 1,800 urine specimens were screened by Gram stain to detect bacteriuria. Pellets of bacteria were obtained by centrifuging specimens containing greater than or equal to 1 gram-negative bacillus of a single morphological type per oil immersion field. Direct susceptibility tests and identifications were performed from pellets by using the AutoMicrobic system (AMS). Results were compared with culture results by routine AMS methods. Of the 145 specimens showing only gram-negative bacilli on Gram stain, 113 grew greater than or equal to 10(5) CFU of a single species per ml. Compared with routine AMS identifications, the direct method correctly identified 105 (92.9%) of the isolates. Identifications were available within 8 h for 77% of the isolates. When compared by MICs, 93.2% of the direct susceptibility test results agreed with routine AMS results within one twofold dilution. Comparisons by category call indicated that overall complete and essential agreements were 89.9 and 97.8%, respectively, with 1.0% very major, 1.0% major, and 8.1% minor errors. Cefamandole and cephalothin had the lowest correlations by both comparisons. Within 8 h, susceptibility results were available for 94.3% of the isolates. This method offers the advantage of rapid detection, prompt processing, and earlier reporting of complete results for positive urine specimens.     

Comparison of microdilution and agar dilution procedures for testing antibiotic susceptibility of Neisseria gonorrhoeae

Studies were run in parallel to compare the broth microdilution method and the chocolate agar dilution method for testing antibiotic susceptibility of Neisseria gonorrhoeae. Six clinically relevant drugs were tested against 23 clinical isolates of N. gonorrhoeae, including several penicillinase-producing, as well as multiply resistant, strains. Results showed that the MIC obtained by the two methods were not significantly different. The microdilution method appears to be a more sensitive system for discriminating penicillinase activity. The microdilution system is a more expedient method for screening new antibacterial agents and is more readily adaptable to new automated equipment.     

Rapid identification and antimicrobial susceptibility testing of gram-negative bacilli from blood cultures by the AutoMicrobic system.

A procedure was developed which allows direct identification and antimicrobial susceptibility testing of fermentative and nonfermentative gram-negative bacilli from positive blood cultures. A 10-ml sample was removed from turbid blood culture bottles, and the bacteria were washed and concentrated by centrifugation. The bacterial pellet was used to inoculate an Enterobacteriaceae Plus Identification Card and a Gram-Negative General Susceptibility Card of the AutoMicrobic system. Results with these cards were compared with results obtained with standard technique for 196 blood cultures seeded with recent clinical isolates. Identification of most cultures was available in 8 h, whereas the antimicrobial susceptibility results were available in an average of 4.7 h for all organisms. Direct identification was correct for 95% of the cultures, whereas the antimicrobial susceptibility data had an average agreement of 87% with 3.8% very major and 1.4% major errors. In using this procedure it was possible to provide accurate preliminary identification and results of antimicrobial susceptibility tests for gram-negative bacilli on the same day that a blood culture was determined to be positive.     

Evaluation of the VITEK 2 system for rapid direct identification and susceptibility testing of gram-negative bacilli from positive blood cultures

This study explores the possibility of combining the BacT/Alert Microbial Detection System with the VITEK 2 system to achieve rapid bacterial identification and susceptibility testing. Direct inoculation of bacterial suspension to the VITEK 2 ID-GNB card and AST-NO09 card was made by differential centrifugation of blood cultures of organisms with gram-negative enteric bacillus-like morphology. A total of 118 strains were investigated; of these, 97 (82.2%) strains were correctly identified to the species level and 21 (17.8%) strains were not identified; by comparing the results with those of the reference method of API identification systems using a pure culture, it was found that no strain had been misidentified. Among the 21 strains with no identification, 13 (61.9%) strains were nonfermenters. The direct-identification reporting time of VITEK 2 was 3.3 h. Direct testing of susceptibility to 11 antibiotics, i.e., amikacin, cefepime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, netilmicin, piperacillin, piperacillin-tazobactam, and tobramycin, was also performed by using the broth microdilution (MB) method according to the NCCLS guidelines as a reference. After comparing the MICs of the VITEK 2 system with those obtained by the MB method within +/-twofold dilution, it was determined that the 1,067 organism-antibiotic combinations had an overall correct rate of 97.6% (1,041 combinations). The rates of susceptibility to the 11 antibiotics ranged from 88.7 to 100%, respectively. Only two (0.2%) and four (0.4%) combinations of the susceptibility tests gave very major errors (i.e., reported as sensitive by the VITEK 2 system but shown to be resistant by the MB method) and major errors (i.e., reported as resistant by the VITEK 2 system but shown to be sensitive by the MB method), respectively. The reporting time for the direct testing of susceptibility against the 11 antibiotics for 97 blood culture isolates by the VITEK 2 system ranged from 3.3 to 17.5 h. Compared with conventional methods that require 1 or 2 days, this method can make same-day reporting possible and thus permit better patient management.     

Comparison of Etest and agar dilution method for antimicrobial susceptibility testing of Flavobacterium isolates.

The Etest was evaluated as a possible alternative to the standard agar dilution method for susceptibility testing of nine antimicrobial agents against Flavobacterium species. In studies of 100 clinical isolates, the agreement between the MICs (+/-1 log2 dilution) obtained by the two methods was acceptable for cefotaxime, ceftazidime, amikacin, minocycline, ofloxacin, and ciprofloxacin (> 90%). Conversely, the agreement between the results obtained for piperacillin was limited (84%). The overall agreement was 92.5%.