Barbara H. Minshew

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

Cation components of Mueller-Hinton agar affecting testing of Pseudomonas aeruginosa susceptibility to gentamicin.

Seven lots of Mueller-Hinton agar were examined for calcium and magnesium contents and their distribution in pools or compartments. Gel disruption and centrifugation yielded the soluble cations, which varied from 9 to 113% of the total calcium and from 76 to 102% of the total magnesium. Throughout the experiments, a standardized disk diffusion test, using Pseudomonas aeruginosa (ATCC 27852) and a 10-μg gentamicin disk, served as an indicator for medium performance. Zone diameters correlated well with the sums of the soluble calcium and magnesium values in the different lots (r = −0.85). Ionized calcium, presumably the biologically active ion, was measured with a calcium-specific electrode. It represented only a fraction of the soluble calcium pool in three lots. Autoclaving resulted in shifts of the cations between the different pools. Addition of magnesium to one medium lot resulted in shifts of soluble and ionized calcium, indicating an interdependence of calcium and magnesium, and zone diameters correlated with soluble magnesium (r = −0.98), soluble calcium (r = −0.96), and ionized calcium (r = −0.96) in this experiment. Manipulation of one medium to match the performance of another showed that excess amounts of both ions were required to obtain similar performance. Satisfactory performance of an individual medium can be obtained by cation supplementation, but simple adjustment will not suffice for all media. The interaction of the other cation pool components must also be evaluated.

Common Plasmid Specifying Tobramycin Resistance Found in Two Enteric Bacteria Isolated from Burn Patients

Tobramycin-resistant burn wound isolates of Klebsiella pneumoniae and Enterobacter cloacae, together with Escherichia coli K-12 transconjugants from these two strains, were examined for plasmid deoxyribonucleic acid (DNA). All the resistant strains contained a common, high-molecular-weight, covalently closed circular DNA plasmid that was absent in the tobramycin-susceptible E. coli recipient strain. The common plasmid residing in E. cloacae was designated pIE098, and that residing in K. pneumoniae was designated pIE099. Both plasmid species were found to have a molecular mass of approximately 60 × 106 daltons and a guanine-plus-cytosine content of 50 mol%. The DNA that was extracted from all of the tobramycin-resistant strains tested was able to hybridize to 86 to 100% with pIE098 and pIE099 [3H]DNA generated by EcoRI to produce fragments of a size similar to those generated by BamHI. This study illustrates the usefulness of simple screening methods for antibiotic resistance plasmids in a hospital epidemiological situation. Images

Resistance of Pseudomonas aeruginosa to Gentamicin and Related Aminoglycoside Antibiotics

This study was undertaken to investigate biochemical, genetic, and epidemiological aspects of resistance to aminoglycoside antibiotics among 650 consecutive isolates of Pseudomonas aeruginosa from Parkland Memorial Hospital, Dallas, Tex. In 364 strains, minimal inhibitory concentrations were 25 μg/ml or greater for gentamicin (G), tobramycin (T) or kanamycin (K). Four patterns of resistance were noted: (A) G, T, K (four strains), (B) G, K (23 strains), (C) T, K (one strain), and (D) K (336 strains). Gentamicin acetyltransferase (GAT) activities were associated with resistance to gentamicin in strains of groups A and B, whereas kanamycin phosphotransferase activity was found in strains of group D. The GAT from group B strains acetylates both gentamicin and tobramycin. Resistance to gentamicin and susceptibility to tobramycin may reflect the fact that the Kms for tobramycin (25 to 44 μg/ml) of GAT activities in these group B strains are much greater than the Kms for gentamicin (1.9 to 2.7 μg/ml) and exceed the minimal inhibitory concentrations for tobramycin (1.25 to 7.5 μg/ml). GAT from strains of group A was associated with resistance to G, T, and K. Gentamicin acetyltransferases can be distinguished by their specificities for aminoglycoside substrates. The substrate specificity of GAT from group B strains is similar to that reported for GATI, but the specificity of GAT from group A strains differs from those described for GATI and GATII. Conjugal transfer of gentamicin or tobramycin resistance from our strains of P. aeruginosa to various potential recipient strains was not observed. Pyocin typing showed that many, but not all, of the strains resistant to gentamicin were similar, and retrospective epidemiological investigation revealed that these strains were isolated almost exclusively from patients in the adult and pediatric burn intensive care units and geographically continguous areas of the hospital.

Effect of Different Lots of Mueller-Hinton Agar on the Interpretation of the Gentamicin Susceptibility of Pseudomonas aeruginosa

Population distributions and quality control data for strains of Pseudomonas aeruginosa tested for gentamicin susceptibility on six lots of Mueller-Hinton agar were analyzed. The lots of agar were used in three University of Washington hospitals from April 1975 through October 1977. The analyses indicated that the performance of members of the P. aeruginosa populations in each hospital closely followed the performance of the quality control strain, P. aeruginosa ATCC 27853, when tested on each lot of Mueller-Hinton medium. The variability of zone diameters with the P. aeruginosa populations and the quality control strain indicated that a fixed indeterminate range (13 to 16 mm) of gentamicin susceptibility was not applicable to these organisms as it was with the Enterobacteriaceae. Variability in gentamicin susceptibility results was demonstrated in both minimal inhibitory concentration and disk diffusion tests when eight selected P. aeruginosa strains and the quality control strain were tested on each lot of medium. This variation in susceptibility to gentamicin was not related to the total Ca2+, Mg2+, or Zn2+ content of each lot of medium. The data demonstrated that a moving indeterminate range of gentamicin susceptibility, 3 to 6 mm below the mean zone diameter of the quality control strain, was a suitable criterion for strains tested on a single medium lot. These results illustrate the importance of defining stringent performance standards for media used in the susceptibility testing of P. aeruginosa with gentamicin and other aminoglycoside antibiotics.

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.

Transferrable Resistance to Tobramycin in Klebsiella pneumoniae and Enterobacter cloacae Associated with Enzymatic Acetylation of Tobramycin

Among gram-negative bacilli isolated from burn wound cultures, some strains of Enterobacteriaceae were resistant to tobramycin (minimal inhibitory concentration [MIC]≥ 20 μg/ml) but susceptible to gentamicin (MIC ≤ 5 μg/ml). One Klebsiella pneumoniae and two Enterobacter cloacae strains were selected for studies on their mechanisms of resistance to aminoglycoside antibiotics. Resistance to high concentrations of tobramycin (MICs of 25 to 50 μg/ml) was conjugally transferred to a susceptible Escherichia coli strain at rates of 1.2 × 10−4 to 2.8 to 10−4 per donor cell, suggesting that resistance is controlled by R factors. Resistances to tobramycin, kanamycin, and neomycin were cotransferred. Enzymatic activities were present that acetylated tobramycin, gentamicin, and kanamycin in osmotic lysates from the donor and transcipient strains. Enzymatic adenylylation of these aminoglycosides was not observed. The aminoglycoside-acetylating activities from K. pneumoniae and E. cloacae resembled kanamycin acetyltransferase (KAT) in their specificity for aminoglycoside substrates. Not all isolates of bacteria that produce KAT are resistant to tobramycin, but the factors that determine susceptibility or resistance to tobramycin in KAT-producing bacteria have not yet been established.

Comparison of a Radioimmunoassay with an Enzymatic Assay for Gentamicin

A radioimmunoassay and an enzymatic assay for gentamicin have been compared. The correlation coefficient for results of gentamicin assays performed by the two methods with 45 serum specimens was 0.90. A similar standard curve for the radioimmunoassay was obtained with gentamicin complex, with gentamicin Cl, Cla, or C2, or with sisomicin as ligand, but tobramycin did not compete with [3H]gentamicin for binding to the antiserum.

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.

Effect of ionized calcium and soluble magnesium on the predictability of the performance of Mueller-Hinton agar susceptibility testing of Pseudomonas aeruginosa with gentamicin.

The soluble and ionized calcium and magnesium contents of 18 lots of Mueller-Hinton agar medium from three different manufacturers were analyzed, and the results were correlated with medium performance. A standardized disk diffusion test, with Pseudomonas aeruginosa (ATCC 27853) and a 10-microgram gentamicin disk, served as an indicator of medium performance. Zone diameters correlated well with the ionized calcium values and the sum of the ionized calcium and soluble magnesium values in the different lots (r = -0.88 for both). Zone diameters correlated poorly with ionized magnesium values (r = -0.57), which were best described by a curvilinear relationship. Supplementation of lots of Mueller-Hinton agar medium with equivalent amounts of calcium and magnesium as the chloride, gluconate, or glycerophosphate salts produced identical decreases in zone sizes. Adjustment of deficient lots of Mueller-Hinton agar medium with ionized calcium or soluble magnesium or both (as the gluconate salts), to match the concentrations in lots that provided satisfactory zone sizes (17 to 19 mm), resulted in performance comparable to that of the control lots. Sixteen strains of Pseudomonas aeruginosa, ranging from resistant to susceptible, responded to cation adjustment in the same manner as the ATCC quality control strain. Satisfactory medium performance can obviously be assured by biological means in aminoglycoside susceptibility testing of Pseudomonas aeruginosa on Mueller-Hinton medium; however, cation adjustment of medium to predetermined levels of ionized calcium and soluble magnesium can now also provide desirable performance levels for P. aeruginosa on Mueller-Hinton medium.