Comparison of the checkerboard and E-test methods used for the analysis of two antibiotics combination

The aim of this study was to compare checkerboard method with E-test assay for interaction analysis of aminoglycosides in combination with other antibiotics on selected clinical bacterial strains. In the first step, MIC values of analysed antibiotics, against particular bacterial strain were established. In the next step, antibiotics interaction was analysed by checkerboard technique and E-test. We found some difficulties while comparing these two methods. The checkerboard and E-test results corresponded in about 55%. Twenty-one percentage of results obtained by both methods showed some discrepancies. In 15% of cases, because of high MICs values, comparison of the results was impossible. Some investigators declare FIC indexes, from over 0.5 up to 4, for neutral effects. Sharing this point of view, above 21% of discrepancies results agreed. In such situation, definite disagreement was observed only in 8% of obtained results. In this investigation, additive and neutral effects were dominant. The E-test technique is less-laborous than standard agar method. In this study the E-test assay indicated synergy in only one case. Because of the manner in which the E-test strips were placed on the agar (scales intersecting at the MICs) only dramatic cases of antagonism were detected. Mild cases were undetected because the inhibition zone run under the crossed strips and was therefore unreadable and interpreted as indifference. On the basis of these results, examination of interactions between antibiotics by E-test, appears to be possible alternative to checkerboard method with mentioned limitations.     

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.     

In Vitro Interactions Between Antifungals and Methotrexate Against <Emphasis Type="Italic">Aspergillus</Emphasis> spp.

Methotrexate has been widely used in the treatment of osterosarcoma, intracranial lymphomas and leukemia. However, patients are also at high risk of opportunist pathogens such as Aspergillus spp. infection for their deeply depressed immunity. The optimal choice of antifungal agents during the infection of Aspergillus for these patients is necessary to be explored. In this study, we investigated the interactions between antifungals and methotrexate against Aspergillus in vitro. A total of 23 clinical isolates of Aspergillus spp. were studied. Microdilution checkerboard technique was performed to evaluate the interaction of methotrexate with voriconazole, itraconazole, terbinafine and amphotericin B. The highest rate of synergy was obtained for the combination of terbinafine and methotrexate, which exhibited synergy against 60.9% (14/23) of strains. No interaction was detected for the combinations of methotrexate plus itraconazole or amphotericin B against 95.7% (22/23) or 100% of strains, respectively. Although voriconazole exhibited indifferent against 87% (20/23) of strains when combined with methotrexate, antagonism effect was found against 13% (3/23) of strains. The positive interactions of terbinafine and methotrexate were also certified by disk diffusion assay. In addition, we observed the morphological changes for the interaction of methotrexate with terbinafine against Aspergillus. Further inhibition and distortion of growth were found after the combination of terbinafine and methotrexate compared with the drugs treated alone. Clinical studies are warranted to further elucidate optimal treatments for the immucompromised patients with Aspergillus infection.     

Interactions of antibiotics and extracts of <Emphasis Type="Italic">Helichrysum pedunculatum</Emphasis> against bacteria implicated in wound infections

The effect of combinations of the crude acetone and aqueous extracts of Helichrysum pedunculatum leaves and eight antibiotics was determined by means of checkerboard and time-kill methods. In the checkerboard method, synergy of 45.8 % was observed, being independent of Gram reaction, with combinations in the aqueous extract yielding largely (18.8 %) antagonistic interactions. The time-kill assay detected synergy (45.8 %) that was also independent of Gram reaction with a potentiation of more than 3 orders of the bactericidal activity of the test antibiotics. The crude leaf extracts of H. pedunculatum could thus be considered to be potential source of a broad-spectrum antibiotic-resistance-modifying compounds.     

Effects of Antifungal Agents Alone and in Combination Against Candida glabrata Strains Susceptible or Resistant to Fluconazole

The rise of Candida spp. resistant to classic triazole antifungal agents has led to a search for new therapeutic options. Here, we evaluated combinations of antifungals in a checkerboard assay against two groups of Candida glabrata strains: one containing fluconazole-susceptible clinical isolates (FS) and another containing fluconazole-resistant laboratory derivative (FR). The most synergistic combination observed was amphotericin B + flucytosine (synergistic for 61.77 % of FS strains and 76.47 % of FR strains). The most antagonistic combination observed was ketoconazole + flucytosine (FS 61.77 % and FR 55.88 %). Surprisingly, most combinations evidenced indifferent interactions, and the best synergism appeared when amphotericin B and flucytosine were combined against both groups of isolates.     

Synthesis and anti Methicillin resistant Staphylococcus aureus activity of substituted chalcones alone and in combination with non-beta-lactam antibiotics

A total of 30 chalcone analogues was synthesized via a base catalyzed Claisen Schmidt condensation and screened for their in vitro antibacterial activity against Methicillin-sensitive Staphylococcus aureus (MSSA) and Methicillin-resistant Staphylococcus aureus (MRSA) alone or in combination with non beta-lactam antibiotics namely ciprofloxacin, chloramphenicol, erythromycin, vancomycin, doxycycline and gentamicin. In the checkerboard technique, fractional inhibitory concentration indices (FICI) show that the following combinations like ciprofloxacin with 25 (4'-bromo-2-hydroxychalcone); doxycycline with 21 (4-hydroxychalcone); doxycycline with 25; and doxycycline with 4 (2',2-dihydroxychalcone) were synergistic against MRSA. In term SAR study, the relationship between chalcone structure and their antibacterial activity against S. aureus and synergy with tested antibiotics were discussed. Possible mechanisms for antibacterial activity of chalcones alone as well as the synergistic effect in combinations were proposed by molecular modeling studies, respectively. Combinations of chalcones with conventional antibiotics could be an effective alternative in the treatment of infection caused by MRSA.     

Comparison of checkerboard and time-kill methods for the analysis of two antibiotics combined

Activity interaction analysis of two antibiotics by two methods: checkerboard and "time-kill" was compared during this study. Combinations of procaine penicillin, polymyxin B and bacitracin with neomycin and procaine penicillin with dihydrostreptomycin were examined. Checkerboard method is the most widely used technique for antimicrobials interactions analyses. The "time-kill" method, performed by the broth macrodilution technique, provides a dynamic picture of antimicrobial action and interaction over time (based on serial colony counts). Differences of "time-kill" method and the checkerboard technique, allow single visual examination (after 16 to 24 hours of incubation). Additive and inhibition effects were observed in combinations of neomycin with beta-lactam antibiotic (procaine penicillin) and peptide antibiotics (bacitracin and polymyxin B) on clinical strain S. Enteritidis IL 35 "Time-kill" method also confirmed observations mentioned above. In combinations of procaine penicillin with dihydrostreptomycin on strains E. coli IL 531 and E. coli IL 256 synergy effects on checkerboard technique were noticed. Such observation was not confirmed by the "time-kill" method. The methodologies and definitions of synergism are variable and not standardized. This situation should be improved, because comparison of the results obtained by different methods becomes a very difficult task.     

Evaluation of the synergistic potential of vancomycin combined with other antimicrobial agents against methicillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus spp strains

Methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococcus spp (CNS) are the most common pathogens that cause serious long term infections in patients. Despite the existence of new antimicrobial agents, such as linezolid, vancomycin (VAN) remains the standard therapy for the treatment of infections caused by these multidrug-resistant strains. However, the use of VAN has been associated with a high frequency of therapeutic failures in some clinical scenarios, mainly with decreasing concentration of VAN. This work aims to evaluate the synergic potential of VAN plus sulfamethoxazole/trimethoprim (SXT), VAN plus rifampin (RIF) and VAN plus imipenem (IPM) in sub-minimum inhibitory concentrations against 22 clinical strains of MRSA and CNS. The checkerboard method showed synergism of VAN/RIF and VAN/SXT against two and three of the 22 strains, respectively. The combination of VAN with IPM showed synergistic effects against 21 out of 22 strains by the E-test method. Four strains were analyzed by the time-kill curve method and synergistic activity was observed with VAN/SXT, VAN/RIF and especially VAN/IPM in sub-inhibitory concentrations. It would be interesting to determine if synergy occurs in vivo. Evidence of in vivo synergy could lead to a reduction of the standard VAN dosage or treatment time.     

特比萘芬与其他抗真菌药物对外阴阴道念珠菌病病原菌的体外相互作用研究

目的 了解特比萘芬( TEB)分别与氟康唑(FLU)、伊曲康唑(ITR)、咪康唑(MCZ)、克霉唑(CMZ)及制霉菌素(NYS)联合对外阴阴道念珠菌病患者分离的念珠菌体外相互作用.方法 受试念珠菌株46株,包括白念珠菌(20株)、热带念珠菌(15株)、光滑念珠菌(4株)、克柔念珠菌(3株)、近平滑念珠菌(3株)和季也蒙念珠菌(1株).方法参考CLSIM27-A3方案中的微量液基稀释法,观察各药单独和联合后的MIC,并计算FICI.结果 TEB、FLU、ITR、MCZ、CMZ和NYS 的单药MIC范围分别为0.5～256μg/mL、0.5～ 128 μg/mL、0.5 ～ 32.0 μg/mL,0.125 ～ 16μg/mL、0.03 ～ 4.0 μg/mL和2～8μg/mL.当TEB与FLU、ITR、MCZ和CMZ联合时,对白念株菌表现为协同作用的分别为100％ (20/20),80％ (16/20),55％ (11/20)和90％ (18/20);对1株季也蒙念珠菌均显示为协同作用;对热带念珠菌,TEB分别与上述4种唑类药物联合时,显示有协同作用的分别为67％ (10/15),60％ (9/15),20％(3/15)和20％ (3/15);TEB与唑类药物联合对光滑念珠菌、克柔念珠菌和近平滑念珠菌均无协同作用;TEB与NYS联合时对所有菌株均无协同作用.结论 TEB与唑类药物联合对不同种的念珠菌显示有不同的效应,但对外阴阴道念珠菌病主要的致病菌白念珠菌显示有良好的协同作用.TEB与NYS联合对念株菌均无协同作用.     

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.     

Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens

This study investigated the antibacterial activities of sophoraflavanone G from Sophora flavescens in combination with two antimicrobial agents against oral bacteria. The combined effect of sophoraflavanone G and the antimicrobial agents was evaluated using the checkerboard method to obtain a fractional inhibitory concentration (FIC) index. The sophoraflavanone G+ampicillin (AM) combination was found to have a synergistic effect against S. mutans, S. sanguinis, S. sobrinus, S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis, whereas the sophoraflavanone G+gentamicin (GM) combination had a synergistic effect against S. sanguinis, S. criceti, S. anginosus, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis. Neither combination exhibited any antagonistic interactions (FIC index >4). In particular, the MICs/MBCs for all the bacteria were reduced to one-half - one-sixteenth as a result of the drug combinations. A synergistic interaction was also confirmed by time-kill studies for nine bacteria where the checkerboard suggested synergy. Thus, a strong bactericidal effect was exerted through the drug combinations, plus in vitro data suggested that sophoraflavanone G combined with other antibiotics may be microbiologically beneficial rather than antagonistic.     

Efficacy of Cryptdin-2 as an Adjunct to Antibiotics from Various Generations Against Salmonella

Emerging drug resistance in Salmonella coupled with the recent poor success rate of antibiotic discovery programs of the pharmaceutical industry is a cause for significant concern. It has forced the scientific community to look for alternative new classes of antimicrobial compounds. In this context, combinations of antimicrobial peptides (AMPs) and conventional antibiotics have gained interest owing to their versatile applications. The present study was therefore planned to evaluate the synergistic effects, if any, of cryptdin-2, a mouse Paneth cell alpha-defensin, in combination with four different antibiotics i.e. ciprofloxacin, ceftriaxone, cefotaxime and chloramphenicol, which are conventionally used against Salmonella. Minimum bactericidal concentrations of the selected antimicrobial agents were determined by micro and macro broth dilution assays. In-vitro synergy between the agents was evaluated by fractional bactericidal concentration index (checkerboard test) and time-kill assay. Cryptdin-2-ciprofloxacin, cryptdin-2-ceftriaxone and cryptdin-2-cefotaxime combinations were found synergistic as evident by in vitro assays. This synergism provides an additional therapeutic choice by allowing the use of conventional antibiotics in conjunction with AMPs against MDR Salmonella.     

Susceptibility to chemotherapeutics and the occurrence of metallo-beta-lactamases in hospital Pseudomonas aeruginosa strains

The aim of the study was evaluation of susceptibility of Pseudomonas aeruginosa strains isolated from patients hospitalized in different wards of Rydygier's Hospital in Krakow in 2005. Bacteria were identified on the basis of typical morphology confirmed by Gram-staining microscopy and by biochemical tests--ID 32 GN strips using ATB system (bioMerieux, France). The susceptibility of all isolates to a panel of antimicrobial agents was performed using disk diffusion method. The highest in vitro activity against clinical strains demonstrated ceftazidime (88.6% of susceptible strains) while the lowest in vitro activity against clinical strains demonstrated imipenem (50.4% of susceptible strains). It was also observed that 40.2%(53) of strains were resistant to meropenem and imipenem. Carbapenem resistant P aeruginosa strains were tested for MBL production. We performed disk synergy test for MBL detection with EDTA, 2-MPA and ceftazidime, imipenem. The presence of a distorted inhibition zone was interpreted as a positive result for MBL production. Positive results of disk synergy tests were confirmed by Etest MBL strips. Metalo-beta-lactamases were detected in 13 isolates resistant to carbapenems.     

Aminoglycoside resistance rates, phenotypes, and mechanisms of Gram-negative bacteria from infected patients in upper Egypt

With the re-emergence of older antibiotics as valuable choices for treatment of serious infections, we studied the aminoglycoside resistance of Gram-negative bacteria isolated from patients with ear, urinary tract, skin, and gastrointestinal tract infections at Minia university hospital in Egypt. Escherichia coli (mainly from urinary tract and gastrointestinal tract infections) was the most prevalent isolate (28.57%), followed by Pseudomonas aeruginosa (25.7%) (mainly from ear discharge and skin infections). Isolates exhibited maximal resistance against streptomycin (83.4%), and minimal resistance against amikacin (17.7%) and intermediate degrees of resistance against neomycin, kanamycin, gentamicin, and tobramycin. Resistance to older aminoglycosides was higher than newer aminoglycosides. The most common aminoglycoside resistance phenotype was that of streptomycin resistance, present as a single phenotype or in combination, followed by kanamycin-neomycin as determined by interpretative reading. The resistant Pseudomonas aeruginosa strains were capable of producing aminoglycoside-modifying enzymes and using efflux as mechanisms of resistance. Using checkerboard titration method, the most frequently-observed outcome in combinations of aminoglycosides with β-lactams or quinolones was synergism. The most effective combination was amikacin with ciprofloxacin (100% Synergism), whereas the least effective combination was gentamicin with amoxicillin (53.3% Synergistic, 26.7% additive, and 20% indifferent FIC indices). Whereas the studied combinations were additive and indifferent against few of the tested strains, antagonism was never observed. The high resistance rates to aminoglycosides exhibited by Gram-negative bacteria in this study could be attributed to the selective pressure of aminoglycoside usage which could be controlled by successful implementation of infection control measures.     

In Vitro Activity of Sodium New Houttuyfonate Alone and in Combination with Oxacillin or Netilmicin against Methicillin-Resistant Staphylococcus aureus

Background

Staphylococcus aureus can cause severe infections, including bacteremia and sepsis. The spread of methicillin-resistant Staphylococcus aureus (MRSA) highlights the need for novel treatment options. Sodium new houttuyfonate (SNH) is an analogue of houttuynin, the main antibacterial ingredient of Houttuynia cordata Thunb. The aim of this study was to evaluate in vitro activity of SNH and its potential for synergy with antibiotics against hospital-associated MRSA.

Methodology

A total of 103 MRSA clinical isolates recovered in two hospitals in Beijing were evaluated for susceptibility to SNH, oxacillin, cephalothin, meropenem, vancomycin, levofloxacin, minocycline, netilmicin, and trimethoprim/sulfamethoxazole by broth microdilution. Ten isolates were evaluated for potential for synergy between SNH and the antibiotics above by checkerboard assay. Time-kill analysis was performed in three isolates to characterize the kill kinetics of SNH alone and in combination with the antibiotics that engendered synergy in checkerboard assays. Besides, two reference strains were included in all assays.

Principal Findings

SNH inhibited all test strains with minimum inhibitory concentrations (MICs) ranging from 16 to 64 µg/mL in susceptibility tests, and displayed inhibition to bacterial growth in concentration-dependent manner in time-kill analysis. In synergy studies, the combinations of SNH-oxacillin, SNH-cephalothin, SNH-meropenem and SNH-netilmicin showed synergistic effects against 12 MRSA strains with median fractional inhibitory concentration (FIC) indices of 0.38, 0.38, 0.25 and 0.38 in checkerboard assays. In time-kill analysis, SNH at 1/2 MIC in combination with oxacillin at 1/128 to 1/64 MIC or netilmicin at 1/8 to 1/2 MIC decreased the viable colonies by ≥2log₁₀ CFU/mL.

Conclusions/Significance

SNH demonstrated in vitro antibacterial activity against 103 hospital-associated MRSA isolates. Combinations of sub-MIC levels of SNH and oxacillin or netilmicin significantly improved the in vitro antibacterial activity against MRSA compared with either drug alone. The SNH-based combinations showed promise in combating MRSA.     

Value Addition in the Efficacy of Conventional Antibiotics by Nisin against Salmonella

Frequent and indiscriminate use of existing battery of antibiotics has led to the development of multi drug resistant (MDR) strains of pathogens. As decreasing the concentration of the antibiotic required to treat Salmonellosis might help in combating the development of resistant strains, the present study was designed to assess the synergistic effects, if any, of nisin, in combination with conventional anti-Salmonella antibiotics against Salmonella enterica serovar Typhimurium. Minimum inhibitory concentrations (MICs) of the selected antimicrobial agents were determined by micro and macro broth dilution assays. In-vitro synergy between the agents was evaluated by radial diffusion assay, fractional inhibitory concentration (FIC) index (checkerboard test) and time-kill assay. Scanning electron microscopy (SEM) was also performed to substantiate the effect of the combinations. In-vivo synergistic efficacy of the combinations selected on the basis of in-vitro results was also evaluated in the murine model, in terms of reduction in the number of Salmonellae in liver, spleen and intestine. Nisin-ampicillin and nisin-EDTA combinations were observed to have additive effects, whereas the combinations of nisin-ceftriaxone and nisin-cefotaxime were found to be highly synergistic against serovar Typhimurium as evident by checkerboard test and time-kill assay. SEM results revealed marked changes on the outer membrane of the bacterial cells treated with various combinations. In-vivo synergy was evident from the larger log unit decreases in all the target organs of mice treated with the combinations than in those treated with drugs alone. This study thus highlights that nisin has the potential to act in conjunction with conventional antibiotics at much lower MICs. These observations seem to be significant, as reducing the therapeutic concentrations of antibiotics may be a valuable strategy for avoiding/reducing the development of emerging antibiotic resistance. Value added potential of nisin in the efficacy of conventional antibiotics may thus be exploited not only against Salmonella but against other Gram-negative infections as well.     

The effects of some formulation factors used in ophthalmic preparations on thiomersal activity against Pseudomonas aeruginosa and Staphylococcus aureus

AIMS: To investigate the effects of formulation ingredients used in ophthalmic preparations on thiomersal activity against Staphylococcus aureus and Pseudomonas aeruginosa. METHODS AND RESULTS: Minimum inhibition concentrations (MICs) of the tested ingredients and their combinations were studied using partial broth dilution checkerboard method. Complex formation was determined using differential scanning calorimetry (DSC) and u.v. scan. Isotonic agents showed insignificant difference in thiomersal activity. Low concentrations of propylene glycol and glycerol (2 to about 6.5%) significantly reduced the activity of thiomersal against P. aeruginosa. Higher concentrations up to about 40%, of the two cosolvents did not affect the antibacterial activity. Viscosity increasing agents significantly reduced the antibacterial activity of thiomersal. Low concentrations of 0.05% and 0.05-0.1% of ethylenediaminetetraacetic acid resulted in a significant decrease in thiomersal activity against S. aureus and P. aeruginosa, respectively. However, concentrations above 0.25 and 0.5 up to 4% caused significant increase on the antibacterial activity against the two later micro-organisms, respectively. CONCLUSIONS: Results showed that thiomersal formed complexes with ingredients containing polyhydroxy groups and chelating agents, thus thiomersal is not recommended to be used with such ingredients. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the importance of proper selection of ingredients and preservatives for ophthalmic preparations.     

粘菌素对耐碳青霉烯类抗生素鲍曼不动杆菌的抗菌作用

感染耐碳青霉烯的鲍曼不动杆菌(CR-Ab)常与高发病率和死亡率相关联,而可供选择的治疗方案有限,大多基于与粘菌素联用。长期用药导致CR-Ab对粘菌素也产生一定抗性。为了评估含有或不含有粘菌素的不同抗菌组合对从CR-Ab感染患者收集的CR-Ab临床分离株的体外抗菌活性,本研究从本院就诊的患者中收集CR-Ab菌株,通过常量肉汤稀释法(MBD)测定最低抑菌浓度(MICs),通过定性(棋盘法)和定量(即杀菌测试)方法评估各组药物协同活性。结果发现所有菌株均是碳青霉烯类抗性的,且其中两株菌对粘菌素有抗性。棋盘法结果表明含粘菌素的组合在不同处理时间下具有完全协同作用,粘菌素+万古霉素和粘菌素+利福平表现出最高的协同增效作用;不含粘菌素的组合则在35.7%的菌株中观察到完全协同作用。杀菌测试表明粘菌素+美罗培南、粘菌素+替加环素和美罗培南+替加环素组合对粘菌素敏感和低粘菌素抗性的菌株具有杀菌和协同作用,而只有粘菌素+万古霉素和粘菌素+利福平组合表现出持久的杀菌活性。     

Detection of the expression of extended-spectrum beta-lactamases (ESBL) in clinical strains of Klebsiella pneumoniae and Escherichia coli: comparison of E-test and two-disc methods

The aim of this study was to compare the effectiveness of E-test and two disc methods applied for the detection of extended spectrum beta-lactamases. All strains were tested by E-test, by double-disc synergy test (DDST) according to Jarlier (cefotaxim, ceftazidim, aztreonam and clavulanic acid) and also by disc test according to Appleton (cefpodoxime and cefpodoxime with clavulanic acid, CPD and CD01 disc). We tested 148 clinical strains of E. coli and 78 strains of K. Pneumoniae. In case of K. pneumoniae, the activity of the ESBLs was detected among 30 strains--both in E-test, Jarlier test and Appleton test. Among E. coli, four strains were found ESBL-positive in the test according to Jarlier but only three strain of these when E-test and Appleton test was used. The results of investigations performed suggest, that E-test and disc methods according both Jarlier and Appleton have the same effectiveness in detection ESBLs among K. pneumoniae strains. However, in case of E. coli, interpretation of results may present a problem.     

Minimal inhibitory concentrations of Candida species to five antifungals by using the reference dilution micromethod and the E-test.

It is accepted that the frequency of candidosis has increased during the last decade, specially in hospitalized patients. The more frequent use of azole antifungals and the recognition of isolates of Candida sp resistant to these and other drugs such as 5-fluorocytosine constitute a great need for a reproducible and useful C. albicans in vitro susceptibility testing method for monitoring antifungal therapy in clinical mycological laboratories. The E-test is a novel agar diffussion technique for testing the susceptibility of yeasts against a defined continous gradient of drug and could be used by most clinical laboratories. In this study the E-test and the NCCLS reference microbroth method (M27-P guidelines) were used to determine the MICs of amphotericin B, 5-flucytosine, itraconazole, fluconazole and ketoconazole for 50 clinical isolates of Candida albicans, Torulopsis glabrata, C. tropicalis and Hansenula anomala and five reference ATCC strains. The main purpose of the study was to compare the results obtained by the two methods. In general good agreement (+/- 1 dilution) was otained between both methods, despite differences observed for some species-antifungal combinations in which the MICs were lower by the E-test than by the microbroth method. MICs for C. albicans and T. glabrata to amphotericin B were < 0.50 microg/mL. Two isolates of C. albicans and two others of H. anomala, showed MIC < 8 microg/mL for 5- flucytosine. All isolates of T. glabrata and 40% of C. albicans showed MICs > 16 microg/mL for fluconazole. The results of this study indicate that E-test is an alternative for susceptibility testing to the NCCLS reference method. Because its simplicity it seems to be an easier test for routine clinical laboratories.