Serial Dilution Tests Research Articles

The in vitro activity of beta-lactam antibiotics against gentamicin and/or carbenicillin-resistant Pseudomonas aeruginosa strains.

We studied the behaviour of 56 clinical isolates of Pseudomonas aeruginosa strains against the following beta-lactam antibiotics: cefotaxime, cefoperazone, cefsulodin, lamoxactam, Ro 13-9904, azlocillin, mezlocillin and ticarcillin. Twenty-six strains were resistant to gentamicin and 30 to gentamicin and/or carbenicillin. The MICs were measured by the serial dilution test on solid agar. Cefsulodin was the most active cephalosporin against carbenicillin-resistant strains (MIC greater than or equal to 128 mg/l); it inhibited 56.6% of these strains at a concentration of 8 mg/l. Azlocillin was the most active penicillin, inhibiting 79.96% of the same strains at a concentration of 64 mg/l. Cefsulodin was the most active cephalosporin against the gentamicin-resistant group of Pseudomonas aeruginosa strains (MIC greater than or equal to 8 mg/l) which were sensitive to carbenicillin (MIC less than or equal to 64 mg/l). It inhibited 100% of the strains at a concentration of 4 mg/l. All of the penicillins studied inhibited all of the strains in this group. The required concentrations were the following: 16 mg/l for azlocillin, 32 mg/l for mezlocillin and 64 mg/l for ticarcillin.

Correlation of Serum Bactericidal Activity with Antimicrobial Agent Level and Minimal Bactericidal Concentration

Few data are available to show how serum dilution test results correlate with results of antimicrobial assays and determinations of minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs). Serum dilution tests were performed for 65 patients with bacteremia from serious infections. Assays for antimicrobial agent levels and determinations fo MICs and MBCs against the infecting organisms were performed simultaneously. Mueller-Hinton broth and Mueller-Hinton broth supplemented with Ca++, Mg++, and pooled human serum were compared as diluents for the serum either medium yielded positive correlations, better correlations were found between measured and expected serum bactericidal activities when both ther serum dilution test dilutions and the MIC and MBC determinations were done in the supplemented MHB-human serum solution.

In vitro studies with Win 42122-2: comparison with gentamicin, netilmicin, and amikacin

The in vitro activity of Win 42122-2 against gram-negative clinical isolates was compared in serial twofold broth dilution tests with gentamicin, netilmicin, and amikacin. Against 173 gentamicin-susceptible Enterobacteriaceae, the activity of Win 42122-2 was generally twofold less than those of gentamicin or netilmicin and similar to that of amikacin. Against 60 gentamicin-susceptible nonfermentative gram-negative bacilli, including P. aeruginosa, the activity of Win 42122-2 was four- to eightfold less than that of gentamicin or netilmicin and two- to fourfold less than that of amikacin. Minimal bacterial concentrations for Win 42122-2 were usually similar to minimal inhibitory concentrations. Win 42122-2 was not highly active against gentamicin-resistant bacteria. Win 42122-2 was as active as gentamicin against Mycobacterium tuberculosis but was less active than gentamicin or amikacin against atypical mycobacteria. Win 42122-2 interacted synergistically with penicillin G against enterococci, including strains highly resistant to streptomycin.

Single-disk diffusion testing (Kirby-Bauer) of susceptibility of Proteus mirabilis to chloramphenicol: significance of the intermediate category.

The significance of the intermediate category of the single-disk diffusion test (Kirby-Bauer) of antibiotic susceptibility has never been clearly defined. Thirty-two percent of 756 clinical isolates of Proteus mirabilis were of intermediate susceptibility to chloramphenicol, a higher percentage than for any other species. The breakpoint separating susceptible and intermediate isolates nearly bisected the frequency distribution of zone diameters of P. mirabilis but not that of the other species. The breakpoint separating susceptible and intermediate isolates nearly bisected the frequency distribution of zone diameters of P. mirabillis but not that of the other species tested. By serial broth dilution testing, the minimal inhibitory concentrations (MICs) of chloramphenicol of 50 individual isolates of P. mirabilis were 3.9 to 22.1 micrograms/ml (geometric mean, 8.0), whereas the MICs of susceptible Escherichia coli, Klebsiella, and Enterobacter strains were 2.0 to 3.9 micrograms/ml (geometric mean, 2.9). Seventy percent of isolates of P. mirabilis with MICs of 7.8 to 15.6 micrograms/ml were classified as susceptible by disk testing. We conclude that existing Kirby-Bauer breakpoints do not accurately discriminate P. mirabilis isolates that are marginally susceptible to chloramphenicol. These data underscore the difficulty of applying a single set of breakpoints to all species and suggest that species-specific breakpoints would more accurately predict the MIC equivalent of given zone diameters.

In vitro synergy and antagonism of antifungal agents against yeast-like fungi.

Some antifungal agents have been tested qualitatively in various associations against species of Candida, Cryptococcus and Torulopsis. The method used was the channel test and the results are confirmed by comparison with the serial dilution test in agar. The antagonism in vitro with the combination of amphotericin B imidazole suggests that caution must be exercised before prescribing the antifungal drugs in combination for man. The frequent synergy of flucytosine with econazole is, however, encouraging because of the low toxicity of the 2 drugs. Under the limited conditions described, the combination of flucytosine and amphotericin B was not found synergistic but additive on the strains of C. albicans used in this study. This combination was found synergistic for a strain of C. parapsilosis and is useful for avoiding resistance to flucytosine.

Serum dilution tests for determination of antibacterial activity

Serum dilution tests for determination of antibacterial activity

Evaluation and relevance of isolated test reactions to cobalt.

During a 5-year period, 286 of 4,034 (7.1%) standard tested eczema patients reacted to cobalt. Fifty (1.2%) showed "isolated" cobalt reactions (i.e. without simultaneous reaction to chromium and/or nickel). A follow-up study of 36 of these patients included a serial dilution test (SDT). Eleven of 15 patients with negative reactions in the SDT had demonstrated weak reactions in the previous standard test, whereas stronger initial reactions had been noted in 19 of 21 patients who reacted positively. Sixteen SDT-positive and six SDT-negative patients were definitely or probably exposed to cobalt in their working or home environments. The majority of patients with "isolated" cobalt sensitivity also had other contact sensitivities (especially to rubber chemicals) or atopic dermatitis. Therefore in some cases of "isolated" cobalt reactions another contact sensitivity or skin disease may be a prerequisite for the development of cobalt allergy. It is however possible that the simultaneous presence of reactivities to different test substances is only a reflection of general skin vulnerability. An uncertainty in testing methodology must also be considered when evaluating test reactions to cobalt. Finally, present results show that cobalt allergy unassociated with contact sensitivity to other substances or other skin diseases is rare.

Strain of Trichomonas vaginalis Resistant to Metronidazole and Other 5-Nitroimidazoles

A strain of Trichomonas vaginalis (IR-78), recently isolated from a patient afflicted with recurrent symptomatic trichomoniasis, showed resistance to metronidazole, tinidazole, and nimorazole in vitro as well as in vivo. In a serial dilution test using cysteine monohydrochloride-peptone-liver infusion-maltose medium, T. vaginalis IR-78 was only resistant under aerobic conditions. Under anaerobic conditions it was as susceptible as the normal reference strain. The minimal lethal concentrations of metronidazole, tinidazole, and nimorazole for IR-78 were 100, 50, and 50 mug/ml aerobically and 0.4, 0.4, and 0.2 mug/ml anaérobically, respectively. The efficacy of metronidazole, tinidazole, and nimorazole was assessed in vivo by oral administration to mice simultaneously infected with IR-78 both subcutaneously and intraperitoneally. The CD(50) (dose needed to cure 50% of infections) of each compound was significantly higher for the subcutaneous than for the intraperitoneal infection. In contrast, there was little difference in CD(50) for these infections in mice inoculated with a susceptible trichomonas strain. The CD(50)'s for all three compounds against intraperitoneal and subcutaneous infections with IR-78 were 2 to >70 times higher than for susceptible strain E. Both forms of infection with IR-78 could always be cured with therapeutically acceptable doses of tinidazole and nimorazole; subcutaneous infections could not be cured with tolerated doses of metronidazole.

Light sensitivity and chromium dermatitis.

In 25 consecutive cases of chromium contact allergy we have made four parallel serial dilution tests and irradiated three of the test rows, two with solar-spectrum-like radiation from a 150 W Xenon lamp (4/5 MED and 4 MED) and one row with long-wave ultraviolet radiation (blacklight) alone. Comparing an unirradiated row (control) and the row irradiated with 4/5 MED, the reactions were significantly more intense in 48% of the cases, i.e. the patients reacted at lower chromium concentrations. In a corresponding comparison with 4 MED a significantly more intense reaction was obtained in 36% of the cases, whereas blackslight showed no significant difference compared with the control row. The most intense reactions were obtained in the cases which were anamnestically photosensitive and had low MED values.

Serum Dilution Test for Bactericidal Activity. II. Standardization and Correlation with Antimicrobial Assays and Susceptibility Tests

The serum dilution test for bactericidal activity is widely used and recommended despite variations in method. A standardized serum dilution test is a necessary prerequisite to meaningful comparisons of results among laboratories and reproduci. bility within laboratories. Therefore, we evaluated both microand macrotechniques for a standardized serum dilution test in which Mueller-Hinton broth supplemented with physiologic concentrations of Mg+ and Ca++ and combined with 50% pooled human serum (MHB-S/HS) was used as the diluent. Serum dilution tests were done with 13 antimicrobial agents against strains of Staphylococcus aureus, Streptococcus sanguis, Streptococcus mutans, Streptococcus faecalis, Pseudomonas aeruginosa, and Serratia marcescens isolated from the blood of patients with bacterial endocarditis or septicemia. The physiologic diluent MHB-S/HS performed like human serum in the serum dilution test and gave results comparable to those obtained by determination of minimal inhibitory and minimal lethal concentrations and assay of antimicrobial agents in test serum. Since the bactericidal end point was defined as '99.9% killing of the initial inoculum of 5 x 105 colony-forming units/ml, similar results were obtained with different subculture techniques. When dilution tests are required to guide therapy of systemic infections, we recommend a standardized method in which MHB-S/HS is used as the diluent.

Serum Dilution Test for Bactericidal Activity. I. Selection of a Physiologic Diluent

with 50% pooled human serum (MHB-S/HS); and pooled human serum alone (HS). Protein binding of 13 antimicrobial agents was determined in MHB-S/5%A, MHB-S/ HS, and HS. Results obtained by macro- and micromethods for broth and serum dilution tests were compared with those obtained by the agar dilution test for carbenicillin, oxacillin, and gentamicin against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Activity of oxacillin against S. aureus was decreased fourfold by the presence of human serum in concentrations of -25%. Physiologic concentrations of Ca++ and Mg++ decreased the susceptibility of P. aeruginosa to gentamicin fourfold. The performance of MHB-S/HS was similar to that of HS, whereas the performance of MHB, MHB-S, and MHB-S/5%A was not similar to that of HS. MHB-S/HS is a suitable diluent for use in standardization of the serum dilution test. The serum dilution test is a direct method of

Routine patch testing with cadmium chloride.

During a 22-month period, 2.0% cadmium chloride in distilled water was included in our routine patch test series. Twenty-five of 1502 eczema patients showed reactions (1.7%), but none had any relevant history. At serial dilution test only one patient reacted to 1.0% cadmium; all the other dilutions applied gave negative reactions. Analysis by atomic absorption spectrophotometry of the cadmium chloride used for patch testing showed that it contained less than 1 microgram/g of chromium, copper, cobalt and nickel. The conclusion is that no certain case of contact allergy to cadmium chloride has been revealed. An experimental study using the guinea pig maximization test is in progress.

Correlation of the Serial-dilution Protamine Sulfate Test with Models of “Hypercoagulability” and Thrombosis in Dogs

In a dog model, combinations of venous-injury thrombosis, homologous serum-induced "hypercoagulability," and fibrinolytic blockade were evaluated to determine the factors involved in the generation of a positive serial-dilution protamine sulfate test. Stasis and operative procedures did not produce a positive test. Small local thrombi induced by electrical current produced a positive test that tended to persist only during the initial formation and propagation of the thrombus, and was blocked by pretreatment with epsilon-aminocaproic acid, suggesting in this model that the test was detecting mainly early fibrin degradation products. Homologous serum infusion without venous-injury thrombosis also produced positive serial-dilution protamine sulfate test results that could not be blocked with epsilon-aminocaproic acid suggesting here that the serial-dilution protamine sulfate test was detecting mainly soluble complexes of fibrin monomer rather than early fibrin degradation products. The serial-dilution protamine sulfate test is therefore capable of detecting both small local thrombi and serum-induced "hypercoagulability" without thrombosis in the dog model.

In-vitro-Empfindlichkeit von Klebsiella-Enterobacter-Stämmen gegen Cefazolin

Investigation of the resistance of 290 strains of the Klebsiella-Enterobacter group (252 Klebsiella and 38 Enterobacter strains) against cefazolin showed that 63% of the Enterobacter strains were resistant and 50% of the Klebsiella strains were sensitive both in the serial dilution test and in the agar diffusion test. A total of 78% were inhibited by 32 mug cefazolin per millilitre. Isolates from the genitourinary tract were significantly more resistant than those from the respiratory tract.

Carfecillin: antibacterial activity in vitro and in vivo.

Carfecillin, the alpha-phenyl ester of carbenicillin, hydrolyses rapidly in the presence of serum or body tissues to liberate carbenicillin but hydrolysis is less rapid in aqueous solution. The activity of carfecillin in antibacterial tests in vitro depends upon the extent of hydrolysis to carbenicillin, and in conventional serial dilution tests carfecillin shows an antibacterial spectrum generally similar to that of carbenicillin due to extensive hydrolysis. However, in tests in which the extent of hydrolysis is reduced, carfecillin displays lesser activity than carbenicillin against gram-negative bacilli and greater activity against gram-positive cocci. In the presence of serum carfecillin is hydrolysed rapidly to carbenicillin and the activity shown is solely that of carbenicillin. Unlike carbenicillin, carfecillin is well absorbed in mice after oral administration, producing significant carbenicillin blood concentrations and the compound is as effective by the oral route in the treatment of various experimental mouse infections as is parenteral carbenicillin.

The serial thromboplastin dilution test: A presumptive test for hypercoagulability

The serial thromboplastin dilution test: A presumptive test for hypercoagulability

Mode of action and quantitative structure-activity correlations of tuberculostatic drugs of the isonicotinic acid hydrazide type.

Quantitative structure-activity studies have been performed for a series of 2-substituted isonicotinic acid hydrazides by correlating electronic, steric, and lipophilic properties of the substituents with the biological activity date (MIC) from serial dilution tests with Mycobacterium tuberculosis (strain H 37 Rv). The reaction rates for the quaternization of 2-substituted pyridines with methyl iodide were also determined. The rate constants show a similar dependence on the steric and electronic effects of the substituents as the antibacterial activities of the corresponding pyridine-4-carboxylic acid hydrazides. The obtained correlations give evidence that the reactivity of the pyridine nitrogen atom is essential for the biological activity of 2-substituted isonicotinic acid hydrazides and seem to support the hypothesis that isonicotinic acid derivatives are incorporated into an NAD analogue.

Antiviral activity of 9-beta-D-arabinofuranosyladenine. V. Activity against intracerebral Vaccinia virus infections in mice.

Tests for susceptibility to ampicillin and carbenicillin were performed with 35 strains each of Klebsiella, Enterobacter, Serratia, and Proteus, 71 strains of Pseudomonas aeruginosa, and 68 strains of enterococci by serial dilution and disk-diffusion tests employing 10(-3) dilutions of overnight cultures as inocula for both. Commercial 10-mug ampicillin and 50- and 100-mug carbenicillin disks, and freshly prepared 10-, 50-, and 75-mug ampicillin and 10- and 50-mug carbenicillin disks were used. Results were displayed as cumulative distribution curves for both minimal inhibitory concentrations and zone diameters, and as scattergrams for correlating them. Differences in susceptibility to the two antibiotics were small for Klebsiella, Enterobacter, and Serratia and large for the others. The freshly prepared and commercial disks of the same content gave comparable zones. There was good correlation of zone diameter with each disk and the minimal inhibitory concentration. Among the ampicillin disks tested, none was useful for Pseudomonas; with the other species, the 10-mug disk, as well as those with higher ampicillin content, could discriminate susceptible from resistant strains. However, only the 75-mug disk selected some Klebsiella strains susceptible to high concentrations. The 50- and 100-mug carbenicillin disks were equally discriminating for most strains, but the higher concentration was more selective for Klebsiella. The 10-mug carbenicillin disk was as effective as the 50- and 100-mug disks for discriminating among Enterobacter, Serratia, Pseudomonas, and Proteus, but not for Klebsiella or enterococci. The 10(-3) inoculum gave zone sizes considerably larger than those reported by other workers who used the standard Kirby-Bauer method.

Susceptibility of recently isolated pathogenic bacteria to trimethoprim and sulfamethoxazole separately and combined.

A number of media and modifications were used in preliminary tests of susceptibility of common pathogenic aerobic bacteria to trimethoprim (TMP) and sulfamethoxazole (SMZ) separately and combined. The effect of varying the density of the lawn for the disk-susceptibility test was also investigated. Various serial dilution tests in liquid and on solid media were also compared, and the effect of varying the size of the inoculum for these tests was investigated. As a result of these tests, done largely on laboratory-maintained strains, a lawn of overnight cultures diluted 10-3 was considered most useful and practical for the disk tests, but the standard dilution specified in the Kirby-Bauer method was generally included for comparison. In the serial dilution tests, the one using the inocula replicator and 10-3 dilution of culture for inoculum on a modified (thymidinefree) Mueller-Hinton agar was selected as optimal, but similar tests with undiluted cultures and other modifications of various media were also used for some species. By use of these methods groups of recently isolated common pathogenic bacteria were tested by the disk and inocula replicator methods for susceptibility to TMP and SMZ separately and combined in ratio of 1:20 in the former and 1:16 in the latter method. Against nearly all strains of almost all species tested TMP alone and combined with the sulfonamide was found to be active in clinically useful concentrations, and against a large proportion of the organisms the two agents were clearly synergistic even for strains that were moderately or highly resistant to SMZ.

In vitro investigations with oxolinic acid, a new chemotherapeutic agent.

The susceptibility pattern against oxolinic acid of 161 bacterial strains causing urinary tract infections was investigated simultaneously in serial dilution and agar diffusion tests. Oxolinic acid proved to be very active against <i>E. coli</i> and pathogens of the <i>Proteus</i> and <i>Klebsiella-Aerobacter</i> group. Most <i>Staphylococcus aureus</i> strains were also sensitive. The antimicrobial activity against <i>Ps. aeruginosa</i> and <i>Enterococci</i>, however, was low. There was good accordance between the results of the serial dilution tests and of the agar diffusion tests (2 <i>μ</i>g sensitivity discs). In addition, the susceptibility pattern of 621 urinary tract pathogens was evaluated in agar diffusion tests. As compared to other widely used antibiotics and chemotherapeutic agents oxolinic acid demonstrated in vitro a favorable antimicrobial activity.