Antibiotic Susceptibility Testing of Anaerobes: Comparison of Three Methods, Including an Automated Micro-method, and Evaluation of the Effect of Different Media

Disc-diffusion, agar-dilution and microdilution methods were compared for testing the antibiotic sensitivity of anaerobic bacteria. Eight antibiotics and 31 test organisms were used. There was a poor correlation between MIC values obtained by the agar-dilution and microdilution procedures. The test organisms showed an apparently increased sensitivity to the antibiotics when tested by the agar-dilution, as compared with the microdilution, procedures. Columbia Blood Agar and Thioglycollate Haemin agar, were evaluated for the disc-diffusion and agardilution procedures. There was a poor correlation between the MIC values and inhibition zone diameters for the two media, with lower MIC values and larger zones on Thioglycollate Haemin agar.     

Enhancement of antibiotic activity against Staphylococcus aureus by exposure to hyperbaric oxygen

Growth of Staphylococcus aureus ATCC 6538P was studied in stationary broth cultures (11 mm deep) exposed to hyperbaric oxygen (100% O₂ at 3 atm absolute). The minimal inhibitory concentration (MIC) of the following antibiotics was determined after exposure to high-pressure oxygen (HPO) for 3, 6, and 12 hr: penicillin, streptomycin, tetracycline, oxytetracycline, kanamycin, and cephalothin. Logarithmic growth during exposure to HPO was retarded 60%. Air at 3 atm absolute did not retard growth. The longer the exposure of tube dilution tests to HPO, the lower the MIC. Regardless of the antibiotic used, MIC values relative to 100% for unexposed controls were similar for given exposures, and averaged 73% after 3 hr of exposure to HPO, 53% after 6 hr, and 34% after 12 hr. Similar enhancement with HPO and an iodophor suggests occurrence of a general phenomenon with antibacterial agents. Although HPO alone is primarily bacteriostatic, combined therapy with antibiotics and HPO may be useful against bacterial infections because the therapeutic effectiveness of a maximal dosage of antibiotic could be increased.     

The drug sensitivity of meningococci and the characteristics of its determination

The results obtained in the study of antibiotic and sulfamide sensitivity of 197 Neisseria meningitidis strains of groups A, B and C, isolated from the spinal fluid and blood of patients with meningococcal infection hospitalized in the 2nd Clinico-Infectious Hospital, Moscow, in 1984-1989 and studied with the use of the disc diffusion method and the method of serial dilutions of antibiotics in solid culture media, are presented. As revealed in this study, N. meningitidis strains retained their high sensitivity to penicillin and ampicillin (MIC50 = 0.016 and 0.032 micrograms/ml respectively). Sensitivity to tetracycline decreased (MIC50 = 0.5 micrograms/ml) and to rifampicin increased (MIC50 = 0.063 micrograms/ml). 48.5% of strains were resistant to streptomycin. In recent years the proportion of N. meningitidis, resistant to sulfanilamide preparations, significantly decreased and MIC50 was equal to 2.5 micrograms/ml in comparison with 5-10 micrograms/ml in the preceding period. The results of testing sensitivity to antibiotics by both methods coincided. Still the disc diffusion method can be used in epidemiological surveillance on meningococcal infection, while for more exact differentiation of N. meningitidis strains the use of the method of serial dilutions is necessary.     

Therapeutic effect of some antibiotics on experimental staphylococcal infection and its correlation with in vitro activity of antibiotics in sub-inhibitory concentration against Staphylococcus aureus strains

The aim of the study was to demonstrate of whether the therapeutic effects of antibiotics depend on their in vitro activity in sub-inhibitory concentrations against staphylococci. Cloxacillin, gentamicin and lincomycin were used in the study. Groups of S. aureus strains, containing 6 strains with similar MIC values each but different sensitivity to sub-inhibitory antibiotic concentrations (sub-MIC) were selected (a total of 36 trains): i. strains increasing their sensitivity to phagocytosis and bactericidal activity of rabbit leukocytes after incubation with an antibiotic in 0.1 MIC concentration, ii. strains with sensitivity to the above factors unaffected by incubation with an antibiotic in 0.5 MIC concentration. The doses of staphylococci causing death of 90-100% of Swiss albino mice 10 days after i.p. infection were determined. The injected doses (LD 90-100) and various doses of antibiotics were used to determine ED50 values as well as the survival rate of the mice with experimental staphylococcal infections after treatment with these antibiotics. It was demonstrated that effective doses (ED 50) of the antiboitics were significantly lower when the antibiotics were administered once to mice infected with strains S. aureus sensitive to sub-MIC concentrations of the investigated antibiotics than for mice infected with strains resistant to their sub-MIC concentrations. Similar correlations were observed in mice which were given the antibiotics several times (for 7 days): the percentage of the surviving mice was higher in the group infected with sub-MIC sensitive strains. The therapeutic effect of cloxacillin, gentamicin and lincomycin demonstrated a significant correlation with the S. aureus strains used to induce the infections and their sensitivity, or lack of sensitivity in vitro, to phagocytosis and bactericdal activity of leukocytes in the presence of antibiotics in sub-MIC concentrations.     

Development of an accelerated method of determining the antibiotic sensitivity of Cl. perfringens type A]

An express method for determination of antibiotic sensitivity in the strains of Cl. perfringens of type A using Soviet dry nutrient media and antibiotics is proposed. The criteria for estimation of the level of the antibiotic sensitivity of the causative agent of gas gangrene in short periods on the basis of comparison of the data of the antibiotic agar diffusion procedure and the antibiotic MIC were worked out. Twelve antibiotics and 45 collection strains of Cl. perfringens of type A were used in the experiment. The antibiotic agar diffusion method with the use of the nutrient media, microbial load and cultivation conditions developed by the authors is recommended for tentative determination of the antibiotic sensitivity in Cl. perfringens of type A for 4 hours. The use of the agar diffusion method and determination of the antibiotic MIC provided complete estimation of tha antibiotic sensitivity of Cl. perfringens of type A within not more than 24 hours.     

Rapid determination of antimicrobial susceptibility patterns ofListeria monocytogenes isolates by the autobac AIS and MIC procedures

A total of 34 isolates ofListeria monocytogenes were tested against ampicillin, cephalothin, chloramphenicol, erythromycin, tetracycline, and penicillin-G using the Autobac 3-h AIS and the Autobac 5-h MIC procedures. The results were compared to susceptibility category interpretations and MICs determined using the Sceptor system. With the Sceptor System, all isolates were interpreted to be moderately susceptible to ampicillin and penicillin-G, and susceptible to the four other antibiotics. With the Autobac AIS, all isolates were interpreted to be susceptible to all the antibiotics except penicillin-G. All but one of the 34 isolates were interpreted to be resistant to penicillin-G with the Autobac AIS test. The remaining isolate was interpreted to be indeterminant. The Autobac AIS test was unsatisfactory for determining the susceptibility ofL. monocytogenes isolates to penicillin-G. The Autobac MIC results correlated well with the MIC results of the Sceptor system provided that the Autobac was programmed as though it were testing enterococci. The Autobac MIC reported penicillin-G MICs in units per milliliter and required the use of a conversion factor to obtain micrograms per milliliter, and did not allow for the testing of erythromycin. The Autobac MIC susceptibility category interpretations must not be used, as they were derived from an outdated susceptibility standard. The Autobac MIC test may be used if the limitations given above are observed.     

The effects of subminimal inhibitory concentrations of beta-lactam antibiotics against Clostridium perfringens.

The effects of subminimal inhibitory concentrations (sub-MIC) of four beta-lactam antibiotics [penicillin-G (PCG), ampicillin (AMP), cephaloridine (CER), cephalothin (CET)] were tested against Clostridium perfringens type A PB6K, after determining the minimum inhibitory concentrations (MIC) of 29 different Clostridium strains. The majority of the strains were sensitive to all beta-lactam antibiotics. Morphological changes, such as filamentous development and lysis, occurred at concentrations considerably lower than the MIC of CER and CET in C. perfringens. Clear cooperation of AMP and CER with rabbit polymorphonuclear leucocytes (PMNL) against C. perfringens was observed. The filamentous bacteria produced as a result of exposure to sub-MIC of each antibiotic, were phagocytosed easily. The ratios between the drug concentrations (microg/ml) at which the morphological changes began to occur, the minimum antibiotic concentrations (MAC), and the MIC values (microg/ml), were calculated. A large ratio indicated a wide range of effective concentrations below the MIC value for the antibiotics.     

Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro

An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable. We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method. There was good agreement in interpretation of results between the two methods for both P. aeruginosa (90%) and B. cepacia complex isolates (70%) with tobramycin and for P. aeruginosa isolates (70%) with ceftazidime. As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method. The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed.     

Cephalothin, a New Cephalosporin with a Broad Antibacterial Spectrum: I. In Vitro Studies Employing the Gradient Plate Technique

Cephalothin is 7-(thiophene-2-acetamido) cephalosporanic acid; it was prepared by N-acylation of the nucleus of cephalosporin C, 7-aminocephalosporanic acid. Cephalothin had a broad spectrum of antibiotic activity that was essentially unaffected by human serum or inoculum level, the activity of penicillinase, or pH variation of the growth medium. In vitro development of resistance by staphylococci could not be demonstrated, but the gram-negative organisms did develop a stepwise type of resistance to the antibiotic. Staphylococci made resistant in vitro to 5-methyl-3-phenyl-4-isoxazole penicillin were also resistant to cephalothin and to 6-(2,6-dimethoxybenzamido) penicillin; however, the mechanism of resistance to each antibiotic may have differed. Some complications involved in the laboratory evaluation methods currently in use in the field of antibiotics are examined.     

Effects of hypothermic kidney preservation on the isolated perfused kidney: a comparison of reperfusion methods

Two isolated-perfused kidney methods were used to study the effects of hypothermic preservation on renal function in dog kidneys. The isolated-machine-perfused kidney (IMPK) used an in vitro perfusion technique--the perfusate was a Krebs-bicarbonate type delivered to the kidney at 37 degrees C by a mechanical pump at a constant pressure (100 mm Hg). The isolated-blood-perfused kidney (IBPK) utilized transplantation of the preserved kidney to the femoral vasculature. Renal function (urine analysis) was determined over a 1-hr reperfusion interval and included GFR (creatinine clearance), urine formation, and Na+ reabsorption. Kidneys preserved for only 24 hr by cold storage in either Collins'--C3 solution or in hypotonic citrate and kidneys hypothermically perfused for 24 hr demonstrated greater retention of renal function when reperfused by blood (IBPK) than with the in vitro perfusate (IMPK). The GFR was reduced by 38-58% when tested with the IBPK, but by 80-90% when tested with the IMPK. Na+ reabsorption was normal (97%) with blood reperfusion but was reduced to 36-50% in cold-stored kidneys and 82% in hypothermically perfused kidneys determined by machine reperfusion (IMPK). However, kidneys perfused for 72 hr demonstrated more similar renal functions when tested by either IMPK or IBPK. GFR was reduced to 20% (IBPK) and 11% (IMPK) and Na+ reabsorption averaged 76-85% (IBPK or IMPK). These results suggest that either reperfusion method is suitable for determining the effects of renal preservation on kidney function in kidneys preserved for 72 hr but, for short-term preserved kidneys (24 hr), the IBPK model may be preferred.     

Regression Curve Analysis of Cephalosporin Activity

Regression lines were calculated for cephalothin, cephalexin, and cephaloridine by relating zone diameters of inhibition to minimal inhibitory concentrations (MIC) obtained in Mueller-Hinton agar and in Trypticase Soy Agar. A regression line was calculated for cephaloglycin by obtaining MIC values in Trypticase Soy Agar at pH 6.6. Regression lines calculated from MIC values in Mueller-Hinton agar were practically superimposable on those based on MIC values in Trypticase Soy Agar. Organisms susceptible by disc testing to cephalothin were usually susceptible to cephalexin and cephaloridine.     

Combination antibiotic therapy in an outbreak of prosthetic endocarditis caused by Staphylococcus epidermidis.

The efficacy of combination antibiotics in vivo and in vitro was studied during an outbreak of prosthetic endocarditis caused by Staphylococcus epidermidis in 10 patients. The epidemic curve suggested that patients were infected at the time of their operation, with an interval from that time until diagnosis of 11 days to 20 months. The overall mortality was 50%. Four of six patients treated with gentamicin in combination with a penicillin analogue, a cephalosporin or clindamycin survived without reoperation. One of four patients survived when treated with regimens that did not include gentamicin. In vitro studies showed a median minimum inhibitory concentration for methicillin of 8.0 microgram/mL, compared with 0.1 microgram/mL for cephalothin, clindamycin and gentamicin, and a synergistic bactericidal effect between gentamicin and methicillin, cephalothin or clindamycin. These data suggest that gentamicin is a valuable component of combination antibiotic therapy in prosthetic endocarditis caused by S. epidermidis.     

Behavior of lambs in rest pens during long-distance transport

The aim of this study was to determine how one group of lambs utilized 2 consecutive rest periods in novel environments with access to food and water that occurred during 22 hr of motor transport. The 18.5 +/- 0.6 kg lambs (n = 15) were transported for 8 hr and then unloaded for a 6-hr rest period. After 6 hr, the lambs were reloaded for another 8 hr of transport followed by a 24-hr rest period. Reloading for a second 8 hr of transport followed the initial rest period. The percentage of lambs engaged in drinking, eating, lying, playing, or "other" was determined at 15-min intervals. During the 6-hr rest period, peak lying behavior occurred during the 2nd and 6th hr of the period. During the first 6 hr of the 24-hr rest period, the percentage of lambs lying increased while the percentage of lambs eating decreased. In addition, the percentage of lambs lying during the first 6 hr of the 24-hr rest period was greater than during the 6-hr rest period. Lying down had a greater priority than eating during the second (24-hr) rest period.     

Reliability of the Etest in light of the correlation between an antibiotic's critical concentration (Cc) and MIC values.

The study relates to the theory of diffusion methods for antibiotic sensitivity testing. The aim of the study was to show the relationship between the antibiotic critical concentration (Cc) and its minimum inhibitory concentration (MIC). The results contribute to the explanation of the Etest's reliability and support the scientific basis for MIC determination using agar diffusion methods. Susceptibility among 90 clinical isolates of 12 common aerobic bacterial species to gentamicin, erythromycin, or oxacillin was assessed using the multidisc method (for Cc), by the agar dilution method (for MIC) and by the Etest. The results of all three methods were statistically compared and found to be closely related. The regression equation for Cc values and MIC was log2(MIC) = 0.99 x log2(Cc)-0.13; r = 0.99; p < 0.05; the regression equation for Cc values and Etest-MIC (Et) was log2(Et) = 0.86 x log2 (Cc)+0.34; r = 0.96; p < 0.05; the regression equation for Etest-MIC values and MIC was log2(MIC) = 1.12 x log2(Et)-0.50; r = 0.96; p < 0.05.     

Evaluation of antifungal pharmacodynamic characteristics of AmBisome against Candida albicans

A liposomal formulation of Amphotericin B (AmBisome), with small unilamellar vesicles containing amphotericin B, shows characteristic pharmacokinetics as liposomes, and in consequence, has different pharmacological activity and toxicity from amphotericin B deoxycholate (Fungizone). In this study, we evaluated the antifungal pharmacodynamic characteristics of AmBisome against Candida albicans using the in vitro time-kill method and murine systemic infection model. A time-kill study indicated that the in vitro fungicidal activities of AmBisome and Fungizone against C. albicans ATCC 90029 increased with increasing drug concentration. For in vivo experiments, leucopenic mice were infected intravenously with the isolate 4 hr prior to the start of therapy. The infected mice were treated for 24 hr with twelve dosing regimens of AmBisome administered at 8-, 12-, 24-hr dosing intervals. Correlation analysis between the fungal burden in the kidney after 24 hr of therapy and each pharmacokinetic/pharmacodynamic parameter showed that the peak level/MIC ratio was the best predictive parameter of the in vivo outcome of AmBisome. These results suggest that AmBisome, as well as Fungizone, has concentration-dependent antifungal activity. Furthermore, since AmBisome can safely achieve higher concentrations in serum than Fungizone, AmBisome is thought to have superior potency to Fungizone against fungal infections.     

Effects of duration, concentration, and timing of ionomycin and 6-dimethylaminopurine (6-DMAP) treatment on activation of goat oocytes

The protocol of ionomycin followed by 6-dimethylaminopurine (6-DMAP) is commonly used for activation of oocytes and reconstituted embryos. Since numerous abnormalities and impaired development were observed when oocytes were activated with 6-DMAP, this protocol needs optimization. Effects of concentration and treatment duration of both drugs on activation and development of goat oocytes were examined in this study. The best oocyte activation (87-95%), assessed by pronuclear formation, was obtained when oocytes matured in vitro for 27 hr were treated with 0.625-20 microM ionomycin for 1 min before 6-hr incubation in 2 mM 6-DMAP. Progressional reduction of time for 6-DMAP-exposure showed that the duration of 6-DMAP treatment can be reduced to 1 hr from the second up to the fourth hour after ionomycin, to produce activation rates greater than 85%. Activation rates of oocytes in vitro matured for 27, 30, and 33 hr were higher (P < 0.05) than that of oocytes matured for 24 hr when treated with ionomycin plus 1-hr (the third hour) 6-DMAP, but a 4-hr incubation in 6-DMAP enhanced activation of the 24-hr oocytes. Goat activated oocytes began pronuclear formation at 3 hr and completed it by 5-hr post ionomycin. An extended incubation in 6-DMAP (a) impaired the development of goat parthenotes, (b) quickened both the release from metaphase arrest and the pronuclear formation, and (c) inhibited the chromosome movement at anaphase II (A-II) and telophase II (T-II), leading to the formation of one pronucleus without extrusion of PB2. In conclusion, duration, concentration, and timing of ionomycin and 6-DMAP treatment had marked effects on goat oocyte activation, and to obtain better activation and development, goat oocytes matured in vitro for 27 hr should be activated by 1 min exposure to 2.5 microM ionomycin followed by 2 mM 6-DMAP treatment for the third hour.     

Novel system for improved control of filamentous microorganisms in continuous culture

The efficiencies of two 24-hr elevated-temperature tests to recover Escherichia coli from estaurine water were compared simultaneously with the 72-hr standard methods procedure of the American Public Health Association (APHA). From 1,710 tubes, E. coli was recovered 222 times in lauryl tryptose medium incubated at 44 +/- 0.2 C for 24 hr, 261 times in an experimental medium incubated at 44.5 +/- 0.2 C for 24 hr, and 257 times by the 72-hr APHA method. The number of false positives enumerated was similar in all three tests. The data indicated that E. coli in raw seawater could be determined in 24 hr without a significant loss of accuracy.     

Daily Changes in in Vitro Spontaneous Dopamine Efflux from the Corpus Striatum of Male Rats

In the present experiment we examined the spontaneous in vitro dopamine (DA) efflux from superfused corpus striatum (CS) of male rats at 3-hr intervals throughout a 24-hr photoperiod (lights on 0500-1900 hr). Maximal mean efflux, and post-superfusion DA concentrations were obtained at 0600 hr. With the exception of 0600 hr, mean efflux was lower during the light compared to the dark phase. Interestingly, the direction of the efflux profiles also varied as a function of time demonstrating increasing, decreasing and relatively stable profiles over the superfusion period. These changes in overall mean efflux, post-superfusion tissue concentration and efflux rate profile direction indicate that circadian processes play a complex role upon the synthesis/release process of DA from the nigrostriatal dopaminergic system that is revealed under the dynamic conditions of in vitro superfusion of isolated CS fragments.     

Daily changes in in vitro spontaneous dopamine efflux from the corpus striatum of male rats

In the present experiment we examined the spontaneous in vitro dopamine (DA) efflux from superfused corpus striatum (CS) of male rats at 3-hr intervals throughout a 24-hr photoperiod (lights on 0500-1900 hr). Maximal mean efflux, and post-superfusion DA concentrations were obtained at 0600 hr. With the exception of 0600 hr, mean efflux was lower during the light compared to the dark phase. Interestingly, the direction of the efflux profiles also varied as a function of time demonstrating increasing, decreasing and relatively stable profiles over the superfusion period. These changes in overall mean efflux, post-superfusion tissue concentration and efflux rate profile direction indicate that circadian processes play a complex role upon the synthesis/release process of DA from the nigrostriatal dopaminergic system that is revealed under the dynamic conditions of in vitro superfusion of isolated CS fragments.     

Daily Variations in the Sensitivity of Soybean Seedlings to Low Temperature

Every 4 hr during a time span of 32 hr and of 76 hr a different group of soybean seedlings [Glycine max (L.) Merr., cv. Corsoy] maintained under a regime of 16 hr of light followed by 8 hr of darkness was exposed to -10°C for 4 min. Extent of sensitivity to low temperature was evaluated approximately 10 days alter exposure to freezing temperature by determining the weight of the plants and chlorophyll content of the cotyledons. The amount of sensitivity to low temperature was related to the time of exposure and displayed a significant 24-hr oscillation. Plants appeared to be least sensitive to cold injury during the late portions of the light span. Plants subjected to water stress were less sensitive to cold and no significant 24-hr oscillation in response to low temperature could be detected by the cosinor method of analysis.