Assay of the Antibiotic Activity of Serum

One of the drawbacks of the "tube dilution" method for the assay of antibiotics in human serum has been illustrated by utilizing serum-sensitive and serum-resistant strains of Escherichia coli. In the case of serum-sensitive strains, it was found that fresh serum alone may account for the same degree of inhibition and thus yield minimal inhibitory concentrations identical to those obtained with serum combined with antibiotics, that is, "simulated" serum assay specimens. This fallacy of the method is discussed with regard to those instances in which laboratories were merely to utilize the patient's own coliform organism as the test organism, or with respect to the assay of, for example, polymyxins, in which inadvertently a R(ough) and therefore, serum-sensitive strain of E. coli were to be used as the indicator organism. It is recommended that serum-resistant laboratory strains of Staphylococcus aureus or E. coli of known antibiotic susceptibility be employed as the test organisms proper in order to circumvent the inherent bactericidal activity of serum.     

Antifungal susceptibility testing of Trichosporon beigelii to imidazole compounds

Twenty-two strains of Trichosporon beigelii have been tested for susceptibility to imidazole compounds. Ten strains were isolated from untreated genital white piedra lesions and 12 were from the same patients following treatment failure with imidazole compounds. Agar dilution and disk elution methods were compared using two media: yeast nitrogen base and antibiotic assay medium 3 (Difco). Antifungal agents tested were econazole, miconazole, ketoconazole, clotrimazole, and amphotericin B in concentrations of 0.0625-32 micrograms/mL. The most consistent results occurred with antibiotic assay medium 3 and the agar dilution method giving minimal inhibitory concentrations between 0.0625 and 0.25 micrograms/mL. Using yeast nitrogen base agar, minimal inhibitory concentrations were higher ranging from 0.0625 to 2.0 micrograms/mL. End points of growth in the disk elution method were not clearly delineated and ranged from 0.0625 to 8.0 micrograms/mL. The distribution of minimal inhibitory concentrations obtained using different media and methods were compared by chi 2 analysis, and the medium was found to significantly change the minimal inhibitory concentrations. There was no difference in the susceptibility of strains of T. beigelii to imidazole compounds whether isolated before or after treatment. It was concluded that in vitro susceptibility of T. beigelii to imidazole compounds did not necessarily predict efficacy in vivo.     

Micromethod (Spot-Plate) Determination of In Vitro Antibiotic Susceptibility

The spot-plate method for determining in vitro antibiotic susceptibility is a modified version of the standard tube dilution technique, with the same principle of twofold dilution. Results of the spot-plate method are compared with those of the standard tube dilution technique. It is proposed that the spot-plate method is an equally accurate, but more rapid and economical, technique for determining in vitro antibiotic susceptibility.     

Tube Dilution Antimicrobial Susceptibility Testing: Efficacy of a Microtechnique Applicable to Diagnostic Laboratories

A microtechnique for determining antibiotic susceptibilities by the serial dilution method was evaluated in a clinical diagnostic microbiology laboratory. As compared with the standard tube method, an agreement of 94% was achieved for determining minimal inhibitory concentration with +/- one tube dilution as the criterion of comparison. The experience with this system suggests that it could easily be incorporated into diagnostic laboratories as a routine procedure.     

Antimicrobial activity of formylchromones: detection by a micro-scale method

We report the antimicrobial activity of formylchromones. These compounds are remote structural analogues of nalidixic acid and quinolone antibiotics, and their activity was investigated by a simple micro-scale method designed for the determination of minimal inhibitory concentrations (MIC) of drug candidates and antibiotics against aerobic bacteria and yeasts. Minimal bactericidal and fungicidal concentrations (MBC and MFC, respectively) were also determined in connection with the MIC determinations. The results obtained were compared with those obtained using classical agar diffusion methodology. In the MIC method, deep-well micro-titration plates are used, covered by silicone sealing mats that allow diffusion of oxygen to the wells. The appropriate broth is pipetted into the wells, followed by a standardized microbial suspension (except for sterile controls) and a dilution series of the test substance or control antibiotic or a mere control solvent. The use of white non-transparent polypropylene plates allows easy visual inspection of microbial growth. For the MBC and MFC methods, samples are taken from all wells that contain a test substance or control antibiotic and do not display growth in the MIC test. The samples are streaked on agar plates, the liquid is allowed to absorb into the agar, and finally the microbes are spread all over the plate with a bent rod. Colony counts are compared with that of the untreated microbial suspension at the beginning of the MIC test. The MIC method is suitable for high-throughput screening.     

Fusidic acid: new opportunities with an old antibiotic.

The extensive foreign experience with fusidic acid prior to its belated introduction to Canada is reviewed. Fusidic acid is a steroid antibiotic with minimal toxic and hormonal effects that is mainly excreted through the liver. It has a predominantly bactericidal action and does not shown cross-resistance with other antibiotics. Since organisms resistant to this drug form easily in vitro when exposed to low concentrations, complementary treatment with another antibiotic may be required in some clinical situations. Although fusidic acid is active in vitro against a number of organisms, to date it has mainly been used to treat serious infections due to Staphylococcus aureus. The agent appears to be particularly valuable in the treatment of bone and joint infections and in pediatric practice. Fusidic acid will soon be available in Canada for both oral and intravenous administration. Attainable antibiotic levels in many tissues and body fluids greatly exceed the minimum inhibitory concentrations.     

A sensitive standardised micro-gel well diffusion assay for the determination of antimicrobial activity

We have developed a highly sensitive micro-gel well diffusion assay for the determination of antimicrobial activity. In essence, the normal radial diffusion type assay was adapted to perform it in a microtiter plate. We compared our micro-gel well diffusion assay to a radial diffusion assay and a microtiter broth dilution method, using gramicidin S as model antibiotic, and Micrococcus luteus as the indicator organism. The micro-gel well diffusion assay was as sensitive as the microtiter broth dilution method, and approximately twice as sensitive as the radial diffusion method. Data analysis to calculate minimum inhibitory concentration, 50% microbial growth inhibition and maximum inhibitory concentration was refined by generating dose-response curves with the software package Prism 3.0 (Graphpad Software Inc.). The minimum inhibitory concentrations, determined by the three methods, were significantly different (P<0. 001), highlighting the limitations involved in comparing data obtained from different methods.     

Detailed Methodology and Implementation of a Semiautomated Serial Dilution Microtechnique for Antimicrobial Susceptibility Testing

The detailed methodology and implementation of a semiautomatic microtechnique for performing serial dilution antimicrobial susceptibility studies are described. Quantitative susceptibility studies to a battery of antimicrobials are performed routinely on all significant clinical isolates. Results are reported as the minimal inhibitory concentration in micrograms per milliliter of broth. Guidelines relating standard doses of antimicrobials with expected blood and urine levels are presented to facilitate the use of the quantitative data. This microtechnique is used to measure serum and other body fluid levels of antimicrobial agents to document the level attained with a specific course of therapy. This technique is highly reproducible and has a high correlation with, and is at least 10 times faster than, standard glass tube techniques.     

Polymerase chain reaction analysis of laboratory generated bioaerosols

The common methods for analyzing bioaerosols are based on maintaining organism viability and quantifying culturability which may result in the underestimation of microbial concentrations. The present study employed a well-developed technique that only requires cellular DNA to identify organisms. Polymerase chain reaction (PCR) was chosen to amplify specific DNA sequence from an organism, to detect and semi-quantify organisms. Suspensions ofFrancisella tularensis were aerosolized in a chamber, and air samples were collected using impingers. Samples were analyzed using limiting dilution PCR, and the results compared with those from a traditional plate counting. Results indicated that the limiting dilution PCR provides a new way to identify and quantify bioaerosols that does not rely on viability and culturability. Therefore, the method would provide a more reliable estimate of airborne bacterial concentrations compared to traditional plate counts.     

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture

Abstract Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h⁻¹ (15% of μ _max). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.     

Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances

The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium being investigated. MIC values are used to determine susceptibilities of bacteria to drugs and also to evaluate the activity of new antimicrobial agents. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. For broth dilution, often determined in 96-well microtiter plate format, bacteria are inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth is assessed after incubation for a defined period of time (16-20 h) and the MIC value is read. This protocol applies only to aerobic bacteria and can be completed in 3 d.     

Campylobacter and fluoroquinolones: a bias data set?

There is no universally accepted standard method for the isolation of Campylobacter spp. and it is considered that currently available isolation media are not yet optimal for the recovery of Campylobacter spp. from a range of sample types. Almost all methods incorporate antibiotics into the isolation media to inhibit growth of other bacteria within the sample. It is established that the incorporation of such antibiotics into isolation media will inhibit the growth of some Campylobacter spp. as well as other bacteria. The results of the use of such suboptimal isolation methods are that the isolates which 'survive' the isolation procedure will be those which: (i) are able to 'out compete' the rest of the bacteria in the sample, i.e. they are able to grow faster; (ii) are resistant to the antibiotics used in the isolation media; and (iii) are randomly selected by the laboratory technician as being a 'typical'Campylobacter spp. It is clear that such a procedure is intrinsically biased and will mean that species resistant to the antibiotics used in the media will be isolated. This introduces real doubt that the bacteria isolated are truly representative of those initially found on the sample. It is also becoming clear that Campylobacter spp. are rather difficult to isolate as pure cultures and many are in fact mixtures of more than one strain. Again this introduces great uncertainty as to the prevalence and distribution of respective species from the different sample types. This is especially true when considering isolation of Campylobacter spp. causing disease in man as there is no certainty that the selected isolate is that which was responsible for disease. The incorporation of antibiotics into the isolation media not only introduces the issue of species bias but perhaps more importantly exposes the Campylobacter spp. to a cocktail of antibiotics thereby providing the potential for them to 'switch on' antibiotic resistance mechanisms. It might be argued that this has always been the case for isolation of Campylobacter spp., however, we know that the antibiotic cocktails used in media over the last 10 years have changed and indeed there was a time when the filtration protocol which didn't use antibiotics was more widely used. As most reports in the literature do not state what methods were used to isolate Campylobacter spp. it is not possible to quantify any relationship between antibiotics used in the isolation media and susceptibility data. An approved method for Campylobacter susceptibility testing was not available until May 2002, all data generated prior to this date will have been generated using non-standard methods. As tremendous variability in the reproducibility data for Campylobacter spp. was observed during the development of the standard agar dilution susceptibility method, data generated with disk diffusion and broth microdilution methods must be considered with caution. It has been shown that, compared with the conventional agar dilution method, the E-test tends to give rise to lower minimal inhibitory concentrations (MICs) for sensitive strains and higher MICs for resistant strains. There are no recommended antibiotic breakpoint concentrations for Campylobacter spp. A breakpoint is used to separate sensitive from resistant strains of bacteria and is thus crucial to any discussion of antibiotic resistance. This discussion is further complicated by introduction of the terms microbiological and clinical breakpoints. While a microbiological breakpoint can be a useful parameter with regard to identifying resistance factors it cannot on its own be used to predict whether that bacteria will respond to treatment from an appropriate antibiotic. Predicting clinical response is a function of the clinical breakpoint which considers the pharmacokinetic profile of the antimicrobial compound, i.e. the concentration of the antimicrobial compound in the body and the MIC. The National Committee for Clinical Laboratory Standards (NCCLS) uses microbiological, pharmacokinetic and clinical data to establish breakpoints, without c and clinical data to establish breakpoints, without such considerations it is not possible to consider what is truly clinically sensitive and resistant. There are no reported studies that have systematically determined appropriate breakpoints for Campylobacter, there are data however, which relate MICs to clinical outcome. It is without dispute that microbiological resistance in Campylobacter spp. occurs as a result of mutation in the gyrA gene with single point mutations most frequently causing a four- to eightfold shift in the MIC. What is also clear is that if a high enough concentration of antimicrobial relative to MIC of the infecting organism can be achieved not only will the parent organism be killed but also the 'resistant' mutant. Considering the above and the concentrations of ciprofloxacin achieved in the gastro-intestinal tract it is not surprising that clinical cure can be demonstrated for organisms with an MIC of 32 microg ml(-1).     

Accuracy of the disk method for determining antimicrobic susceptibility of common Gram-negative bacilli

A standardized disk susceptibility test was evaluated by comparing results with minimal inhibitory concentrations obtained with agar dilution methods. The agar overlay method was used to test 152 Gram-negative bacilli against eight different antimicrobial agents. One to 3% of the isolates were resistant to an antimicrobic by the MIC method, but appeared to be susceptible by the disk method. Most very major discrepancies involved disk tests withProteus sp., a microorganism notoriously difficult to test reproducibly.Serratia sp. vs. the polymyxins andKlebsiella sp. vs. nitrofurantoin accounted for most other major discrepancies. With other microorganism-drug combinations, the disk test was a reasonably accurate technique for classifying bacteria into resistant or susceptible categories. Gentamicin disk tests were unsatisfactory, but when an intermediate zone category of 13–16 mm was applied, the false susceptible test results were reduced to 2.6%. Intermediate zone sizes were obtained with 6% of the disk tests; most of those isolates were resistant or susceptible but not intermediate in susceptibility. About 11% of the strains had intermediate MICs (5–20% with different drugs), but most of those strains were fully susceptible by the disk technique. *** DIRECT SUPPORT *** A01R4011 00007     

Antibiotic Sensitivities of Organisms Isolated from Mastitic and Nonmastitic Mammary Secretions

Antibiotic sensitivity tests to determine the minimal inhibitory concentrations (MIC) and the minimal lethal concentrations (MLC) for six antibiotics were conducted against mastitic isolates of staphylococci in skim milk and broth. The MIC and the MLC for all the antibiotics except benzylpenicillin were considerably higher in skim milk than in broth. Benzylpenicillin was the most effective antibiotic tested in either medium, and dihydrostreptomycin was the least effective against the organisms tested.     

In vitro activity of a combination of colimycin and tetracycline againstPseudomonas aeruginosa andEscherichia coli

Summary Thein vitro activity of a combination of colimycin and tetracycline againstPseudomonas aeruginosa andEscherichia coli was studied with the aid of tube dilution tests,Elek andHilson's modification of the replica technique, and growth inhibition curves. The interaction between the two antibiotics was as a rule of an additive nature, but a slight synergism was observed in some instances. The values of the minimum inhibitory concentrations did not seem to influence the nature of the interaction. A considerable synergism observed in all tube dilution tests withE. coli strains is attributed partly to addition and partly to suppression of the growth of more colimycin-resistant bacteria by the tetracycline. Possiblein vivo applications of this antibiotic combination are briefly discussed.     

Interlaboratory variability of disc diffusion and agar dilution susceptibility tests with cefamandole and cephalothin

Reproducibility of antimicrobic susceptibility tests was estimated by examining control data accumulated during a multicenter study for evaluating cefamandole and cephalothin. The precision of agar dilution minimal inhibitory concentrations was compared with the standardized Bauer-Kirby disc method. Regression lines were established for each antimicrobic and were used to calculate the range of minimal inhibitory concentration values that corresponded to the observed ranges in zone sizes, thus permitting a comparison of the two types of procedures. The precision of the disc method was equal to or greater than that of the agar dilution method.     

Rapid Sulfonamide Disc Sensitivity Test for Meningococci

Minimal inhibitory concentrations (MIC) of 90 strains of Neisseria meningitidis were determined by a plate dilution technique that employed twofold changes in concentrations of sulfadiazine. The geometric mean of three MIC determinations on each strain was correlated with inhibition zones produced by a 300-mug sulfathiazole disc. The linear relationship between the logarithm of the geometric mean MIC values and the zone diameters was highly significant. Strains were separated into sensitive and resistant populations by both test procedures. Quantitative criteria for interpreting the sensitivity of a strain by the disc test were established.     

Statistical inference for serial dilution assay data

Serial dilution assays are widely employed for estimating substance concentrations and minimum inhibitory concentrations. The Poisson-Bernoulli model for such assays is appropriate for count data but not for continuous measurements that are encountered in applications involving substance concentrations. This paper presents practical inference methods based on a log-normal model and illustrates these methods using a case application involving bacterial toxins.     

Effect of Different Media on In Vitro Studies of Antibiotic Combinations

In an effort to determine the adequacy of a standard broth medium in the evaluation of antibiotic combinations, 20 strains of various bacterial species were studied simultaneously in Mueller-Hinton broth and in freshly drawn human serum from apparently healthy volunteers. Studies of growth dynamics by use of the usual plate dilution technique for quantitating colony-forming units were performed with strains of Staphylococcus aureus (methicillin-resistant and methicillin-susceptible), Streptococcus faecalis, Escherichia coli, Aerobacter, Klebsiella, and Proteus mirabilis. A variety of different antibiotics were investigated. With 19 of the 20 strains, interpretations of synergism or antagonism were the same in both media. Therefore, despite minor variations when the same strain was studied in both serum and broth, it is concluded that Mueller-Hinton broth is an adequate medium for use in studies of chemotherapeutic combinations in vitro. A simplified method for studying bactericidal activity is described, which is deemed practical for clinical microbiology laboratories and which led to the same conclusions regarding the combinations as were obtained by the more arduous plate dilution test.     

A new antimicrobial susceptibility testing method of Escherichia coli against ampicillin by MSPQC

A new antimicrobial susceptibility testing method by multi-channel series piezoelectric quartz crystal (MSPQC) was proposed. This method was used to test susceptibility of clinical Escherichia coli isolates against ampicillin. Both the minimum inhibitory concentrations (MICs) and interpretive categorization of clinical E. coli isolates were determined by proposed method. Comparing tests were run at the same time by the agar dilution method and the disk diffusion method. The experimental results showed that MSPQC method had a good agreement with the reference methods. Compared with those methods, the MSPQC method is simple, rapid, and convenient to perform. It can offer both a minimum inhibitory concentration (MIC) and an interpretive category result.