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.     

In Vitro Activity of Doxycycline Against Bacteria from Clinical Material

Doxycycline (alpha-6-deoxy-oxytetracycline) was tested against various bacteria of recent clinical origin with 30-mug discs. The antibiotic susceptibility of these bacteria to commonly used antimicrobial agents was also established. Those bacteria which responded with equivocal zones of inhibition about the tetracycline compounds were tested by the tube dilution technique. Staphylococci and enterococci consistently displayed greater in vitro susceptibility to doxycycline than to tetracycline or demethylchlortetracycline.     

Diversifications in the Tube Dilution Test for Antibiotic Sensitivity of Microorganisms

The tube dilution method of performing antibiotic sensitivity tests is commonly employed as an accurate method for defining the minimal inhibitory concentration in relation to pathogenic organisms. It is also used as a reference for comparing minimal inhibitory concentrations with the size of the zone of inhibition in the agar diffusion test. Although surveys have shown that there is no standardized method and technique of performing the tube dilution test, it is generally assumed that all of the diversified methods will yield the same results and interpretations. With the assistance of five experts, seven different tube dilution methods were compared; 16 antibiotics, and three organisms for each antibiotic, were used. The conclusions drawn are that, although the accuracy of a single method within its own confines is acknowledged, the minimal inhibitory concentrations and interpretations cannot be interpolated from one laboratory to another where a different technique is employed. The results are frequently discrepant. It is suggested that a uniform method be developed and promulgated for general use.     

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.     

益生乳酸菌的纸片扩散法药敏性试验评价

乳酸菌菌株的药敏性评价是目前国际上益生乳酸菌安全性评价的首要内容, 然而用于乳酸菌药敏性测试的相关国际通引标准还未出台。本文就药敏试验使用的培养基进行了探讨, 并从中选取了合适的商品化培养基——RCA用于乳酸菌的药敏性测试。选取的培养基适合目前市场上常用益生菌的生长, 经方法优化后可得到满意的结果, 为今后制定针对乳酸菌的抗菌药物敏感性试验执行标准提供了一定依据。随后采用纸片扩散法对分属于4个属的9株乳酸菌进行了19类51种抗生素的敏感性测定, 较全面地了解了这些菌株的药敏谱。     

Susceptibility of multiresistant strains of Burkholderia cepacia to honey

Twenty strains of Burkholderia cepacia, isolated principally from the sputum of cystic fibrosis patients, were tested for their susceptibility to eight antibiotics with a modified Kirby-Bauer Disc diffusion technique. All strains exhibited multiple but not identical patterns of antibiotic resistance. The sensitivity of all strains to honey was assessed with an agar dilution method. All strains exhibited susceptibility to concentrations of honey below 6% (v/v). This suggests that honey may have a potential role in the clinical management of B. cepacia infections.     

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.     

Medium-Dependent Activity of Gentamicin Sulfate Against Enterococci

Routine disc diffusion susceptibility tests (Bauer-Kirby technique), employing 5% sheep blood-Mueller-Hinton agar and 10-mug gentamicin sulfate discs, disclosed that a significant number of clinical enterococcal isolates were sensitive to the antibiotic, as also revealed by the agar dilution technique. With few exceptions, the isolates proved resistant to this antibiotic when tested for susceptibility in Brain Heart Infusion and Trypticase soy broth or agar. The addition of 5% sheep blood to Trypticase soy and Brain Heart Infusion agars resulted in markedly enhanced activity of the antibiotic, indicating medium-dependent activity of gentamicin against enterococci. Human serum and urine failed to support optimal growth of enterococci. Thus, it was not possible to correlate the activity of gentamicin in any of the media examined with that in serum or urine.     

Standardization of hyphal growth inhibition rate as a means of evaluating Microsporum spp. in vitro susceptibility to terbinafine, griseofulvin, and ciclopiroxolamine

Reference methods for antifungal susceptibility tests recommend the use of conidia as inoculum. However, some isolates produce few conidia, while the invasive form of filamentous fungi in general is hyphae making susceptibility tests infeaseble. These facts suggest that other than conidia broth dilution method is required for susceptibility tests. The aim of this study was to clarify if the hyphal growth inhibition rate could be used as a method of determining the antifungal susceptibility of genus Microsporum. For this reason, a method which traces hyphal tips automatically and measures their growth rate was standardized for Microsporum spp. Control growth curves and test growth curves obtained by real-time observation of the hyphae groups responses to different concentrations of terbinafine, griseofulvin, and ciclopiroxolamine were used to compare with minimum inhibitory concentrations (MICs) obtained by conidia broth microdilution method. A visible reduction in the growth inhibition rate was observed when hyphal activity was evaluated using the third or fourth serial two-fold dilution below the MIC determined by broth microdilution for terbinafine and ciclopiroxolamine. For griseofulvin, this reduction occurred after the fifth dilution below the MIC. This study highlights the importance of the inoculum type used to determine the in vitro susceptibility of Microsporum strains. We conclude that measurement of hyphal growth inhibition, despite being time consuming, could be a suitable method for evaluating antifungal susceptibility, particularly for fungi as Microsporum spp. that produce a small (or not at all) number of conidia.     

Flow cytometric determination of Enterococcus spp. susceptibility to four antimicrobial agents

Two Enterococcus strains (E. faecalis and E. faecium) isolated from 2 patients in an intensive care unit (blood and drain, respectively) were analyzed for susceptibility to 4 antibiotics (penicillin, vancomycin, gentamicin, streptomycin) by agar dilution standard method (MICs), time-kill and flow cytometry. We compared the data from classical methods of antibiotic susceptibility detection, that are compulsory 24 hrs long and flow cytometry results at 5 and 24 hrs cultivation. The results from both classical and flow cytometric analyses were highly cogent and revealed the fact that flow cytometry is very useful in early diagnosis of bacterial resistance to antibiotics.     

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.     

Influence of Serum and Calcium on the Bactericidal Activity of Gentamicin and Carbenicillin on Pseudomonas aeruginosa

Because calcium was found to be antagonistic in vitro to the activity of colistin and polymyxin B on Pseudomonas aeruginosa, the effects of calcium and serum on gentamicin and carbenicillin were also examined. Serum was antagonistic to gentamicin in antibiotic tube dilution tests on five strains of P. aeruginosa. Serum was not antagonistic to carbenicillin in tube dilution tests. Physiologic concentrations of calcium antagonized the activity of gentamicin but not carbenicillin. The antagonism observed with gentamicin was less than that previously seen with colistin. The antagonistic effect of calcium and serum was removed by a chelating agent. Gentamicin and carbenicillin may be more active in vivo against P. aeruginosa than colistin or polymyxin B.     

The antimicrobial activity of substances derived from the lichens Physcia aipolia, Umbilicaria polyphylla, Parmelia caperata and Hypogymnia physodes

In this study, in vitro antimicrobial activity of the physodic acid, usnic acid, atranorin and gyrophoric acid isolated from the lichens Hypogymnia physodes, Parmelia caperata, Physcia aipolia and Umbilicaria polyphylla, has been investigated. An antibiotic assessment was done against six bacteria (three Gram-positive and three Gram-negative) and eight fungi by determining the minimal inhibitory concentration (MIC) by the broth tube dilution method. The tested lichen substances inhibited growth of all the tested microorganisms. The bacteria showed a higher sensitivity against the tested fungi. The highest antimicrobial activity was found in the usnic acid of the Parmelia caperata lichen, where the lowest MIC was 0.0037 mg/ml against the Klebsiella pneumoniae (even lower than the one given by the streptomycin standard). The weakest antimicrobial activity was found in the physodic acid, which inhibited most of the microorganisms in the concentration of 1 mg/ml. Generally, all the components had relatively strong antimicrobial activity against the tested microorganisms, among which were human and animal pathogens. This could be of significance for their use for pharmaceutical purposes.     

A broth-disc technique for the assay of antibiotic synergism.

A broth-disc technique has been developed to demonstrate the antibiotic synergism of some broad-spectrum penicillins plus an aminoglycoside against Pseudomonas aeruginosa and enterococci. Utilizing the zone sites obtained from Kirby-Bauer susceptibility tests, approximate minimum inhibitory concentrations of each antibiotic upon individual isolates were derived using standard regression curves. One-fourth of each value was calculated and the approximate desired concentration then prepared in Mueller-Hinton broth through use of the labelled antibiotic disc content. Tubes were inoculated with a standardized suspension of organism and incubated at 35 degrees C for 18-24 h. Subsequently, inhibitory end points were recorded. This approach to synergy testing showed good correlation with the standard checkerboard method. The broth-disc synergistic assay is also reproducible, financially feasible, and less time-consuming to execute.     

利用胞内代谢物变化预测大肠埃希菌耐药性

目的利用胞内代谢物量的差异达到预测大肠埃希氏菌的药物敏感性结果。方法收集临床分离大肠埃希菌120株,在头孢他啶存在条件下培养4 h。收集菌体,测定细胞内化合物然后进行多变量分析。结果 120株大肠埃希菌的聚类分析结果很好地体现了药物敏感性特征,57株耐药菌中,有5株被误判,准确率为91%;47株中敏菌株中,有8株被误判,准确率为83%;16株敏感菌中有2株被误判;准确率为88%。结论该方法能够有效预测菌株的药敏结果,而且快速可靠。     

Susceptibility testing of anaerobic bacteria: A review of current methods and future prospects

The current NCCLS document, M11 A2, describes two methods for susceptibility testing of anaerobic bacteria. The reference method utilizes an agar dilution procedure, which is labor intensive and not convenient for testing individual patient isolates. The broth microdilution method does not support the growth of 15–40% clinical isolates and demonstrates poor correlation with the reference method for some members of the Bacteroides fragilis group with β-lactam agents and clindamycin. Etest is a new technique that incorporates an antibiotic gradient onto a plastic strip and utilizes agar media. This method is easily performed, permits growth of all anaerobes, and provides quantitative MICs for rapidly growing strains after overnight (20 hr) incubation. This method is convenient and reliable and enables the laboratory to provide the clinician with MIC data for individual patient isolates within a clinically relevant time period.     

显齿蛇葡萄提取物体外抑菌试验的研究

使用药敏纸片法、试管二倍稀释法测定了显齿蛇葡萄对大肠杆菌、金黄色葡萄球菌、鸡沙门氏杆菌、粪肠球菌、阴沟肠杆菌等5种细菌的抑菌效果,结果表明:显齿蛇葡萄的根、茎、幼叶提取物对5种细菌均有抑菌作用,以幼叶提取物抑菌效果最强,说明显齿蛇葡萄具有广谱高效的抑菌作用,但不同器官的抑菌作用有较大的差别。     

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.     

Determination of tin in biological samples using gaseous hydride generation-inductively coupled plasma-atomic emission spectrometry

A highly sensitive method determining for sub-microgram/gram levels of tin in biological samples is described. Tin hydride reduced by sodium borohydride and trichloroacetic acid solution was introduced into inductively coupled plasma after separation of liquid and excess hydrogen by an improved gas/liquid separator, and emission intensity was measured at a wavelength of 189.989 nm. Samples were decomposed by a nitric acid-perchloric acid mixture and analyzed after dilution by a standard addition technique. The relative standard deviation was 1.2% for a 10 ng/ml tin standard solution with a detection limit of 30 pg/ml.     

Antimicrobial and antiviral screening of novel indole carboxamide and propanamide derivatives

A few series of indole derivatives were screened for antimicrobial, antifungal and anti-HBV activities. The compounds were tested for their in vitro antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and for their antifungal activity against Candida albicans using a disc diffusion method, which measures the diameter of the inhibition zone around a paper disc soaked in a solution of the test compounds. The antimicrobial activity results showed that all compounds are as a active as the standard compound ampicillin against Staphylococcus aureus. It was also found that indole carboxamide derivatives, substituted at 3-position with several benzyl groups, showed better inhibition of Bacillus subtilis than their congeners substituted at 2-position. Activity patterns of the compounds against Escherichia coli and Staphylococcus aureus were found slightly different by the same method. In this case, there was no correlation between structure and activity of the compounds. The antifungal activity of carboxamide derivatives was found higher compared to that of the propanamide derivatives. The minimum inhibitory concentration (MIC) values of some indole derivatives were also determined by the tube dilution technique. The MIC values of the compounds were found nearly 20- to 100-fold smaller compared to the standard compounds ciprofloxacin and ampicillin (1.56-3.13 microg/ml and 1.56-12.5 microg/ml, respectively) against Staphylococcus aureus, Bacillus subtilis and Escherichia coli. The MIC values of the tested compounds showed that these are better inhibitors for Candida albicans. Indole derivatives were screened by the anti-HBV susceptibility test. No compound showed good inhibition against the HBV virus.