Macrodilution Test Research Articles

WIN 57273 is bactericidal for Legionella pneumophila grown in alveolar macrophages.

The in vitro antimicrobial activity of WIN 57273, a new quinolone antimicrobial agent, was determined for 21 Legionella strains, using broth macrodilution and agar dilution testing methods; ciprofloxacin and erythromycin were tested as well. Three different buffered yeast extract media were used for the agar dilution studies, two of which were made with starch rather than charcoal. Broth macrodilution susceptibility testing was performed with buffered yeast extract broth and two Legionella pneumophila strains. Antimicrobial inhibition of L. pneumophila growth in guinea pig alveolar macrophages was also studied, using a method able to detect bacterial killing. The MICs for 90% of the 21 strains of Legionella spp. grown on buffered charcoal yeast extract medium were 0.125 microgram/ml for WIN 57273, 0.25 microgram/ml for ciprofloxacin, and 1.0 micrograms/ml for erythromycin. These MICs were falsely high, because of inhibition of drug activity by the medium used. Use of less drug-antagonistic, starch-containing media did not support good growth of the test strains. The broth macrodilution MICs for two strains of L. pneumophila serogroup 1 were less than or equal to 0.03 microgram/ml for WIN 57273 and ciprofloxacin and 0.125 microgram/ml for erythromycin. WIN 57273, ciprofloxacin, and erythromycin all inhibited growth of L. pneumophila in guinea pig alveolar macrophages at concentrations of 1 microgram/ml, but only WIN 57273 prevented regrowth or killed L. pneumophila after removal of extracellular antimicrobial agent.

Enterococci highly resistant to penicillin and ampicillin: an emerging clinical problem?

Sixteen clinical isolates of ampicillin-resistant enterococci (ARE) were recovered from the microbiology laboratory of a 450-bed rehabilitation medical center from January 1981 to September 1987. These isolates were detected when a disk diffusion test using 10 micrograms of ampicillin on a blood agar plate revealed no zones of inhibition. Tube macrodilution tests yielded an MIC of greater than or equal to 16 micrograms of ampicillin per ml. None of the isolates were penicillinase producers by the chromogenic cephalosporin disk test. Ten isolates were Enterococcus faecium, four isolates were E. raffinosus, one isolate was E. gallinarum, and one isolate was not identified (lost). There were 6 male and 10 female patients. The sources of isolates were urine (n = 7), wound (n = 5), ascitic fluid (n = 2), blood (n = 2), peritoneal catheter tip (n = 1), Bartholin's cyst abscess (n = 1), rectal swab (n = 2), and pancreatic abscess (n = 1). The organism was isolated from multiple sites in 4 patients, was a pure culture isolate in 5 patients, and was part of a polymicrobial flora in 11 patients. Six patients were diabetic, and four had liver cirrhosis. All but four patients had received at least one antibiotic within 3 weeks of ARE isolation. The MICs (micrograms per milliliter) for 50 and 90% of isolates tested, respectively, were as follows: ampicillin, 64 and 64; penicillin, 128 and greater than 128; vancomycin, 1 and 2; gentamicin, 4 and 16; ciprofloxacin, 1.6 and 3.2; imipenem, 128 and greater than 128; and daptomycin (LY146032), 1.6 and 6.4. ARE may be an emerging pathogen in the hospitalized patient population.

In vitro susceptibilities of yeasts to a new antifungal triazole, SCH 39304: effects of test conditions and relation to in vivo efficacy

We used six candidal strains (two Candida albicans and one each of four other species) to study the effects of test conditions on the activity of SCH 39304 compared with that of fluconazole in broth macro- and microdilution assays. Increasing the inoculum from 10(2) to 10(5) yeasts per ml raised the MICs for all isolates up to greater than 512-fold. In contrast, results with a 50% turbidimetric endpoint (50% inhibitory concentration; IC1/2) varied no more than twofold. Similar effects were seen with fluconazole, and both drugs were found to have an associated delay in onset of action. Acidity was found to increase both MICs and IC1/2s. Other effects were observed among four synthetic media, but a consistent pattern was not identified. Incubation temperatures of 37, 35, and 30 degrees C yielded equivalent results. Broth microdilution IC1/2s against most of 40 isolates of C. albicans were 0.31 microgram/ml +/- fourfold for SCH 39304 and 0.16 microgram/ml +/- twofold for fluconazole. Treatment of experimental candidiasis in rats with SCH 39304 and fluconazole resulted in 50% effective doses of 0.33 and 0.49 mg/kg per day, respectively. In contrast, another C. albicans isolate, previously identified as resistant to other azoles, had IC1/2s of 20 micrograms of SCH 39304 per ml and, in vivo, a 50% effective dose of 2.25 mg/kg per day. We conclude that the in vivo efficacy of SCH 39304 correlates with MIC results when broth macrodilution testing is performed with a small inoculum and with IC1/2 results which are independent of inoculum size.

Resistance to ticarcillin-potassium clavulanate among clinical isolates of the family Enterobacteriaceae: role of PSE-1 beta-lactamase and high levels of TEM-1 and SHV-1 and problems with false susceptibility in disk diffusion tests.

Thirty-four clinical isolates of the family Enterobacteriaceae from the University of Texas M. D. Anderson Cancer Center appeared resistant to ticarcillin-potassium clavulanate in agar dilution and broth macrodilution tests. Among those isolates producing a single non-class I beta-lactamase, resistance was due to production of high levels of TEM-1, SHV-1, or class IV enzymes. In five Escherichia coli isolates, production of low levels of PSE-1 was responsible for resistance which seemed due to rapid hydrolysis of ticarcillin rather than diminished susceptibility of PSE-1 to inhibition by potassium clavulanate. Comparisons of dilution and disk diffusion tests revealed major discrepancies, with 65% false susceptibility in the disk test. Revision of the interpretive criteria used for disk diffusion tests from less than or equal to 11 to less than or equal to 18 mm for resistance is proposed to resolve these discrepancies until clinical data are obtained which can be used to determine which in vitro test is most predictive of therapeutic outcome. These new criteria would diminish false susceptibility without introducing false resistance.

Aminoglycoside resistance among Pseudomonas aeruginosa isolates with an unusual disk diffusion antibiogram.

In recent years, a number of clinical microbiology laboratories have isolated Pseudomonas aeruginosa with the unusual aminoglycoside disk diffusion result of resistance to both amikacin and gentamicin but susceptibility to tobramycin (ArGrTs). A total of 39 isolates of P. aeruginosa reported to have this resistance pattern were retested by the standard National Committee for Clinical Laboratory Standards disk diffusion procedure; 30 strains (77%) were confirmed to be ArGrTs. These 30 isolates were further examined for susceptibility to those aminoglycosides by agar dilution and broth micro- and macrodilution methods. Only 27, 27, and 23% of the isolates appeared to be ArGrTs by agar, broth microdilution, and broth macrodilution testing, respectively. Most of the remaining isolates were resistant to all three aminoglycosides when tested by broth dilution and resistant only to gentamicin when tested by agar dilution. The percentages of strains resistant to any particular aminoglycoside by agar dilution, broth microdilution, and broth macrodilution, respectively, were 43, 80, and 70 for amikacin, 97, 93, and 100 for gentamicin, 100, 100, and 100 for netilmicin, 30, 87, and 93 for sisomicin, and 13, 57, and 50 for tobramycin. These results indicate that strains showing the unusual aminoglycoside antibiogram are less susceptible to aminoglycosides in general and should probably be considered borderline resistant to all aminoglycosides. The efficacy of aminoglycosides in the treatment of infections produced by these strains is unknown.

Failure to demonstrate a consistent in vitro bactericidal effect of trimethoprim-sulfamethoxazole against enterococci.

Controversy exists as to the in vitro and in vivo activities of trimethoprim-sulfamethoxazole (TMP-SMX) against enterococci. In this study, we investigated the in vitro activity of TMP-SMX in the type of Mueller-Hinton broth previously reported to give the lowest MICs and MBCs with enterococci. In all instances, MICs were less than or equal to 0.5 microgram/ml. The majority of tests showed MBCs of greater than 32 micrograms/ml, although there was some effect from varying the inoculum and the length of incubation after subculturing. Minor differences were noted when tests were repeated and between the results from microdilution and macrodilution tests and those obtained by the time-kill method. These results, as well as other reports, suggest that TMP-SMX should not be considered a reliable bactericidal agent against enterococci.

Occurrence and reproducibility of the “skip” phenomenon in bactericidal testing of Staphylococcus aureus

A “skip” phenomenon, in which subcultures to determine bactericidal endpoints show discordant results, can make bactericidal testing of Staphylococcus aureus difficult to interpret. Either a single or several consecutive concentrations may be skipped, with insignificant growth from these concentrations, but heavy growth from higher antimicrobic concentrations. Replicate macrodilution minimum bactericidal concentration testing of cephalothin against S . aureus was investigated to determine the frequency and reproducibility of the skip phenomenon in this test. In preliminary testing, the skip phenomenon occurred at 24 hr in 11 of the 30 tests performed on ten S . aureus strains. Incubation of tubes for 48 hr before subculture markedly reduced the number of tests containing a skip tube (four of 30). The skip phenomenon was not reproducible either within concurrent replicate testing or on repeat testing of the same isolate on subsequent days. The ten strains were retested in replicative trials using the macrodilution test comparing Mueller-Hinton broth (MHB) and MHB supplemented with Tween 80. The number of skips occurring in the supplemented broth test was the same (two of 22) at 24 hr as nonsupplemented broth. For reproducible results, macrodilution minimum bactericidal testing requires performance of the test in duplicate, with repeat subculture at 48 hr if the skip phenomenon occurs in both trials at the 24-hr subculture. Killing curves performed on these isolates did not appear to demonstrate any relationship between the skip phenomenon and the paradoxical effect described by Eagle.

European Borrelia burgdorferi isolated from humans and ticks culture conditions and antibiotic susceptibility

Growth of Borrelia burgdorferi in a modified Kelly-medium is described. Borrelia strains were isolated from patients (n = 11) and ticks I. ricinus (n = 19). The modified medium which contained Co-trimoxazole is a very effective medium for isolating and culturing of Borrelia sp. The susceptibility of 7 strains of B. burgdorferi to antibiotics was studied by the macrodilution and microdilution test. After preliminary testing for optimal conditions, we determined MICs in modified Kelly medium. The MIC concentration of each antibiotic was determined as the lowest concentration which completely inhibited growth of the tested organism. The B. burgdorferi was most susceptible to Erythromycin with MIC of less than or equal to 0.15 microgram/ml. Of the Penicillins tested, Ampicillin and Mezlocillin were more active than Penicillin G. The use of Tetracycline-HCl is recommended because of its low MIC in vitro its extra- and intracellular efficiency.

Antibacterial activity of roxithromycin: a laboratory evaluation.

Roxithromycin, formerly known as RU 28965 (9-[O [(2-methoxyethoxy)methyl]oxime]-erythromycin), is a novel 14 atom-membered semisynthetic macrolide with an antibacterial spectrum directed towards Gram-positive cocci and bacilli, Gram-negative cocci and some Parvobacteriaceae. The in vitro antibacterial activity of roxithromycin was compared with those of erythromycin and spiramycin against 275 clinical isolates by using 2-fold broth macro-dilution tests. The antibacterial spectrum of roxithromycin and erythromycin were qualitatively comparable (including the bacteriostatic type of activity and the profile of resistance), but minimal inhibitory concentrations of erythromycin were generally one half those of roxithromycin, except for Corynebacterium sp. and Bacteroides fragilis against which the new macrolide was more active. On the other hand, roxithromycin exhibited a superior in vivo antibacterial activity in laboratory animals, being up to six times more potent than erythromycin in curing experimentally infected mice. Roxithromycin showed high blood levels and long half-lives of elimination in rodents after oral administration, and its bioavailability amounted to 72% in mice and 85% in rats, compared to less than 10% for erythromycin. Roxithromycin was widely distributed throughout the body with a high degree of penetration into all tissues, particularly in the lungs.