Rapid Gentamicin Bioassay Using a Multiple-Antibiotic-Resistant Strain of Klebsiella pneumoniae

A synergistic in vitro bactericidal effect of combinations of chloramphenicol and beta-lactams on strains of Enterobacteriaceae is described. The synergism is seen with strains which are resistant to the beta-lactam and is due to chloramphenicol-induced inhibition of beta-lactamase production.The increasing resistance of gram-negative bacilli to anti-bacterial agents (6), including gentamicin (12) and the cephalosporins (19), raises the possibility of having to resort to a synergistic combination of drugs in the therapy of infections caused by these bacteria.Synergism between chloramphenicol and several beta-lactam antibiotics on strains of Klebsiella-Enterobacter-Serratia, which are resistant to the beta-lactam alone, has recently been described as a not uncommon event (13). The present study was undertaken to examine the occurrence of this synergism with gram-negative bacilli belonging to other genera, and to elucidate its mechanism.MATERIALS AND METHODS Bactericidal effect of combinations of chloramphenicol and three beta-lactams. Ninety-four strains of Enterobacteriaceae isolated in the diagnostic laboratory of the Department of Clinical Microbiology, Hadassah University Hospital, from sputum, infected wounds, stools, and blood cultures were studied. These strains were identified according to Ewing (5) as Escherichia coli (15 strains), Shigella (15 strains), Citrobacter (3 strains), Salmonella (12 strains), Proteus (13 strains), Providence (6 strains), Klebsiella (11 strains), Enterobacter (9 strains), and Serratia (10 strains). The cellophane transfer technique of Chabbert (1) as modified by Cluzel et al. (4) was used to determine a synergistic bactericidal effect. The principles and techniques of this method have recently been described and illustrated in detail (3,7,8,16). The results were interpreted according to previously reported criteria and were classified as synergism, antagonism, or indifference (3,7,8,16). The beta-lactams used were ampicillin, cephaloridine, and carbenicillin.Mechanism of synergistic effect. For these experiments the following strains of beta-lactamase-producing Enterobacteriaceae were used: Enterobacter cloacae NCTC 10005, Klebsiella pneumoniae K 1296 obtained from John Matsen, University of Minnesota (14), and eight strains (see Table 2) isolated from clinical material at the Hadassah University Hospital and identified according to Ewing (5). Each strain was tested for beta-lactamase production by the iodometric method as described by Workman and Farraz (22).Susceptibility to single antibiotics. The susceptibility of these strains to chloramphenicol and cephaloridine was determined by preparing doubling dilutions of the antibiotic in Trypticase soy broth (TSB; Difco). The inoculum was 0.1 ml of a suitably diluted overnight culture in TSB to give a final concentration in the test dilutions of approximately 105 bacteria/ml. The minimal inhibitory concentration (MIC) was expressed in terms of the lowest concentration of antibiotic that totally inhibited visible growth after 24 h...

Section: Methodsmentioning

confidence: 99%

Mechanism of Chloramphenicol-Cephaloridine Synergism on Enterobacteriaceae

Michel

Bornstein

Luboshitzky

et al. 1975

“…These methods include microbiological assays (1,8,10,11), enzymatic assays (4,5,12,13), and radioimmunoassays (7,9). Each type of assay has its own particular advantages.…”

mentioning

confidence: 99%

“…The [3H]gentamicin was purified by chromatography on Sephadex G-10 as described previously (9). The purified [3H ]gentamicin contained 17.8% of the radioactivity in the initial sample and had a specific activity of 0.8 mCi/mg based on determinations of gentamicin concentration by microbiologic (8) (3). After 1 h, 160 mg of gentamicin sulfate was added.…”

mentioning

confidence: 99%

Comparison of a Radioimmunoassay with an Enzymatic Assay for Gentamicin

Minshew

Holmes

Baxter

1975

A radioimmunoassay and an enzymatic assay for gentamicin have been compared. The Within the last few years a number of methods have been developed for measuring the concentration of gentamicin in serum and other biological specimens. These methods include microbiological assays (1, 8, 10, 11), enzymatic assays (4, 5, 12, 13), and radioimmunoassays (7, 9). Each type of assay has its own particular advantages. Enzymatic assays and radioimmunoassays are highly specific and are therefore particularly useful for measuring gentamicin in the presence of other antibiotics. Microbiological assays are inexpensive and can be performed in most diagnostic laboratories without special equipment, but they are limited in specificity by the antibiotic susceptibilities of the test organisms. In the present study, a radioimmunoassay for gentamicin is compared with the enzymatic assay previously used in this laboratory. In addition, published observations concerning the specificity of radioimmunoassays for gentamicin have been extended (7, 9).Reagents for radioimmunoassay of gentamicin were prepared by the following methods. Gentamicin sulfate was radioisotopically labeled with tritium by Amersham/Searle (Des Plains, Ill.). The [3H]gentamicin was purified by chromatography on Sephadex G-10 as described previously (9). The purified [3H ]gentamicin contained 17.8% of the radioactivity in the initial sample and had a specific activity of 0.8 mCi/mg based on determinations of gentamicin concentration by microbiologic (8) and enzymatic (5) assays. Throughout this paper IPresent address:

“…During the past several years a variety of methods have been developed to determine gentamicin levels in serum and other body fluids by more rapid and sensitive methods. Such methods include microbiological assays (1,7,11,14,16,17,21), enzymatic assays (4,5,13,19,20; R. V. Case, Natl. Meet.…”

mentioning

confidence: 99%

Evaluation of the Bactec serum gentamicin assay

Manos¹,

Jacobs²

1979

A radiometric assay for gentamicin was compared with an established radioimmunoassay protocol. The coefficients of variation for within-run, day-to-day, and overall runs were consistently higher for the radiometric method as compared with the radioimmunoassay. The coefficient of correlation for 84 patient sera tested by the two methods for gentamicin levels was 0.82. Though less precise, the radiometric method was felt to be an acceptable means of determining gentamicin levels in laboratories where the radioimmunoassay is unavailable.Gentamicin has established itself as an excellent antimicrobial agent for the treatment of lifethreatening gram-negative infections. The range between therapeutic efficacy and toxicity for gentamicin, however, is narrow, and serum levels must be carefully monitored. The classical procedures for determining adequate antibiotic therapy require 1 to 2 days for results and are fraught with significant error. During the past several years a variety of methods have been developed to determine gentamicin levels in serum and other body fluids by more rapid and sensitive methods. Such methods include microbiological assays (1,7,11,14,16,17,21), enzymatic assays (4,5,13,19,20; R. V. Case, Natl. Meet. Am. Assoc. Clin. Chem., 28th, Houston, Tex., abstr no. 000, 1976), and radioimmunoassays (RIAs) (10,12,13).A rapid radiometric technique for determining serum gentamicin levels has been developed by Johnston Laboratories with their Bactec instrument. The system was originally developed for routine monitoring of blood cultures and has been described elsewhere (2,3,6,9,15,18). The instrument has also been adapted for definitive identification of Neisseria gonorrhoeae and N. meningitidis and is currently being developed for rapid identification and antibiotic susceptibility patterns of Mycobacterium sp. and the determination of certain aminoglycoside levels in patient sera.The principle of the Bactec procedure is based on the fact that gentamicin inhibits the synthesis of urease, an inducible enzyme produced by a species of Proteus. The amount of urease produced is measured indirectly by the amount of "4CO2 released. The quantity of gentamicin in a sample is inversely proportional to the volume of 04CO2 detected by the instrument. The sensitivity range for this assay, according to the manufacturer, is from <1 to 14 ,ug of gentamicin per mi.This study evaluated Johnston Laboratories' gentamicin kit by using the Bactec model 460 to determine gentamicin levels in patient sera as compared with the Monitor Science RIA procedure used routinely in our laboratory. MATERIALS AND METHODSGentamicin test solutions. Two gentamicin test solutions were prepared, containing a low level of gentamicin (3.5 1g/ml) and a high level of gentamicin (10.5 ,ug/ml). The two gentamicin test solutions were prepared by diluting with sterile deionized water a gentamicin (14.0,ug/ml) standard in water provided by the manufacturer. The two test solutions were then aliquoted into 10 vials each and stored frozen at -20°C till assaye...