John C. Sherris

Laboratory evaluation of a rapid, automatic susceptibility testing system: report of a collaborative study.

Seven laboratories participated in a collaborative study to evaluate the Autobac 1 system. Results obtained with this assay system were compared to those obtained by the standardized Bauer-Kirby disk diffusion test, and each of these two methods was compared to the agar dilution technique. Comparison of the Autobac 1 and the disk diffusion results from the seven laboratories showed an overall average of 91.5% interpretive agreement with the 17 antimicrobial agents tested. The distribution in the levels of Autobac 1/disk diffusion agreement was such that with 13 antimicrobial drugs agreement was 90% or higher; with three, between 85 and 90%; and with one, 77% (nitrofurantoin). Comparison of the Autobac 1 and disk diffusion tests with the International Collaborative Study agar dilution test showed that both methods gave levels of agreement with the International Collaborative Study agar dilution technique that were generally high and equivalent. The average overall agreement between the agar dilution test and each of the other two methods was approximately 90%. Disagreements that did occur tended to involve organisms that were drug susceptible by the Autobac 1 system but intermediate or resistant by the other two methods. This was in part due to the narrow intermediate interpretive zone of the Autobac 1 test. In reproducibility studies with the Autobac 1 and disk diffusion methods, no significant differences were observed between the interpretive reproducibility of the two methods. Images

Susceptibility of Enterobacter to Cefamandole: Evidence for a High Mutation Rate to Resistance

Cefamandole minimum inhibitory concentrations (MICs) of 10 strains of Enterobacter were determined by the ICS agar dilution and broth dilution procedures. Agar dilution MICs ranged from 1 to 8 μg/ml, with an inoculum of 104 organisms/spot. Broth dilution MICs were consistently higher, with an inoculum of approximately 7 × 105 organisms/ml. Seven strains showed MICs of ≥64 μg/ml. There was a marked inoculum effect in broth, and skipped tubes were often observed. Variants resistant to 32 μg/ml or more were isolated by direct selection and were shown to occur at a frequency of approximately 10−6 to 10−7. A mutant showing a 16-fold increase in agar dilution MIC was also isolated by indirect selection. These variants and others isolated from broth in the presence of cefamandole were tested for ability to inactivate the antibiotic, using both a biological and a chemical procedure. Two distinct classes of variants were seen. Twelve of 28 were shown by both methods to inactivate the antibiotic, whereas the others, including the indirectly selected mutant, did not. The wild types were also negative by both tests. The higher cefamandole MICs of Enterobacter in broth, thus, appeared to reflect a high frequency of resistant variants that were not detected with the inoculum and end point criteria usually used in agar dilution methods. The ability of some variants to inactivate cefamandole may have resulted from a mutation that extended the activity of Enterobacter cephalosporinase to include this antibiotic.

Studies on the interaction between serum bactericidal activity and antibiotics in vitro.

The possibility of an in vitro interaction between the bactericidal activity of fresh normal human serum and the antibiotics polymyxin B, ampicillin, and kanamycin was investigated

Mutational Enzymatic Resistance of Enterobacter Species to Beta-Lactam Antibiotics

Mutants with enhanced β-lactam resistance were selected from strains of Enterobacter cloacae and E. aerogenes by using three antibiotics. High-level β-lactamase-producing mutants had similar degrees of increased resistance, enzyme substrate profiles, and isoelectric (pI) values irrespective of the selective agent. Reverse mutants from a resistant E. cloacae mutant regained the susceptibility pattern originally exhibited by the wild type, or were of enhanced susceptibility, and no longer expressed increased β-lactamase production. β-Lactamases of the mutants were similar in pI values to the wild-type enzyme. The increased resistance of the mutants therefore appeared to be accounted for by increased β-lactamase production. Images

Relationship of Early Readings of Minimal Inhibitory Concentrations to the Results of Overnight Tests

Broth dilution minimal inhibitory concentration (MIC) readings were compared after different incubation periods and with different inoculum concentrations. The purpose was to determine the best conditions for obtaining early results as close as possible to overnight readings. Initially, 76 antibiotic-organism combinations were tested using the International Collaborative Study technique and inoculum and were read after 3, 8, and 18 h of incubation. Approximately 28% of tests showed fourfold or greater increases in MICs after 18 h of incubation compared with the 3-h readings. No overnight MICs were lower than early readings. MICs of single antibiotics against seven organisms were also read with an automatic particle counter to confirm the validity of the visual readings. Experiments were made to determine whether inoculum manipulation could reconcile the differences between 3- and 18-h MIC results. One hundred and eight organism-antibiotic combinations were tested comparing 3-h MIC readings using an inoculum of 107 organisms per ml with overnight readings using 105 per ml. In 71 cases, readings with both inocula were within the range tested and 57 (86%) were within ±1 log2 of each other and followed an approximately normal distribution. Improved comparability between early read and overnight MICs thus may be achieved by inoculum manipulation, and this may be a suitable approach in the future development of automated procedures.

Emergence in a Burn Center of Populations of Bacteria Resistant to Gentamicin, Tobramycin, and Amikacin: Evidence for the Need for Changes in Zone Diameter Interpretative Standards

From July 1974 through June 1976, a number of isolates of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa from the Burn Center exhibited a shift to smaller zone diameters with gentamicin than did isolates from the general hospital population. Although many had zone diameters ≥13 mm and would have been considered susceptible by this breakpoint, they were found to have minimal inhibitory concentrations (MICs) of ≥8 μg of gentamicin per ml by agar dilution testing. Zone diameters and MICs of gentamicin, tobramycin, and amikacin were subsequently compared for 168 isolates from both the Burn Center and general hospital. The results revealed many isolates that fell into presently used gentamicin- and tobramycin-“susceptible” categories by disk diffusion tests but were resistant by MIC. The data indicated that criteria for gentamicin disk diffusion testing should include an intermediate or indeterminate category, and that the limits of the intermediate category for tobramycin and amikacin should be expanded.

Laboratory determination of antibiotic susceptibility to ampicillin and cephalothin.

With very few exceptions, one of the characteristics of clinically useful chemotherapeutics* has been a high level of in vitro activity against certain pathogenic organisms, and it is in the treatment of infections with such strains that the agents have proved to be clinically useful. Thus, in. vitro measurements of microbial susceptibility are used as initial screening procedures for new agents, to define the spectrum of activity of those that are to be subjected to clinical trial, and to test individual isolates of species from which resistant strains and variants have emerged under the selective pressure of use of the chemotherapeutic. The broad spectrum penicillins and cephalosporins are no exception in these regards, and we shall be describing illustrative experiments bearing on some aspects of the methodology and interpretation of routine tests for the bacteriostatic activity of these agents, with particular reference to our own experience with a single-disc diffusion technique.

Influence of Different Media and Bloods on the Results of Diffusion Antibiotic Susceptibility Tests

As part of an International Collaborative Study, the influence of medium and certain medium components on the results of disc susceptibility testing with nine antibiotics was examined and statistically evaluated. Four basic media, Trypticase Soy Agar, Grove and Randalls formula # 9 agar, and Mueller-Hinton agar with and without 1 mg of l-tryptophan per 100 ml, were used with six bacterial strains. Significant differences in zone diameters occurred with most antibiotics. The largest consistent differences were seen with tetracycline, and appeared to result from varying degrees of chelation of the antibiotic with free cations in the media. Reproducibility studies on different batches of Mueller-Hinton agar from two manufacturers showed some statistically significant differences, which were small except with tetracycline. Reproducibility between the products of a single manufacturer was excellent. The results of experiments to determine the effect of the addition of 5% blood of different species to agar medium showed that medium containing citrated or defibrinated horse, rabbit, sheep, or human blood yielded closely similar zone diameters, except that tetracycline zones were larger with citrated blood. A single type of medium should be selected for routine susceptibility tests whenever possible, and reproducibility of performance of the products of different manufacturers should be sought.

In vitro response of Enterobacter to ampicillin.

Three strains of Enterobacter were studied for their response to ampicillin. They exhibited a basic level of resistance that depended on the medium used and high-level mutational resistance at a frequency of 10(-5) to 10(-7). Two classes of mutants were selected, one of which showed markedly enhanced antibiotic inactivation as indicated by a biological assay and the other of which resembled the wild type in this regard. Both mutants showed cross-resistance to other beta-lactam antibiotics. The results explained discrepancies between traditional broth dilution minimum inhibitory concentration tests and early read automated procedures.

Interpretive Criteria for Cefamandole and Cephalothin Disk Diffusion Susceptibility Tests

A multi-center study of 1,838 clinical isolates established the accuracy of diffusion susceptibility tests with 30-μg cephalothin disks and 30-μg cefamandole disks. The same interpretive zone standards can be applied to tests with either disk but the two drugs cannot be tested interchangeably.