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Nele Wellinghausen - One of the best experts on this subject based on the ideXlab platform.
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Comparison of direct disk diffusion and standard microtitre Broth Dilution susceptibility testing of blood culture isolates.
Journal of medical microbiology, 2020Co-Authors: Andreas Edelmann, Tim Pietzcker, Nele WellinghausenAbstract:Bloodstream infections are life-threatening conditions which require timely initiation of appropriate antimicrobial therapy. The accuracy of direct disk diffusion susceptibility testing of positive blood cultures was investigated, including for the first time beta-lactam/beta-lactam-inhibitor combination antibiotics. Results of direct testing, following the guidelines of the Clinical and Laboratory Standards Institute, were compared to standard microtitre Broth Dilution susceptibility testing of the subcultured isolate on the Merlin MICRONAUT system. Altogether, 758 isolates and 4930 organism/antibiotic combinations from 590 patients were evaluated. With regard to Gram-positive cocci (n=532), agreement between both methods was found in 93.9% of cases, with 1.6% very major, 1.1% major and 2.6% minor errors. For Gram-negative rods (n=226), agreement was found in 91.9% of cases, with 1.2% very major, 0.7% major and 6.3% minor errors. When applying the breakpoints of the Deutsches Institut für Normung for interpretation of MICRONAUT tests, agreement of direct disk diffusion with standard testing decreased to 82.4% in Gram-negative rods, with 3.6% very major, 0.5% major and 13.4% minor errors. A high rate of disagreement was observed with oxacillin and gentamicin in Gram-positive cocci, and with cefuroxime, amoxycillin/clavulanate and piperacillin/tazobactam in Gram-negative rods. In conclusion, the limitations of direct disk diffusion testing of positive blood cultures must be kept in mind by the clinical microbiologist and should, where necessary, be communicated to the clinician to ensure adequate treatment of severely ill patients.
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comparison of direct disk diffusion and standard microtitre Broth Dilution susceptibility testing of blood culture isolates
Journal of Medical Microbiology, 2007Co-Authors: Andreas Edelmann, Tim Pietzcker, Nele WellinghausenAbstract:Bloodstream infections are life-threatening conditions which require timely initiation of appropriate antimicrobial therapy. The accuracy of direct disk diffusion susceptibility testing of positive blood cultures was investigated, including for the first time β-lactam/β-lactam-inhibitor combination antibiotics. Results of direct testing, following the guidelines of the Clinical and Laboratory Standards Institute, were compared to standard microtitre Broth Dilution susceptibility testing of the subcultured isolate on the Merlin MICRONAUT system. Altogether, 758 isolates and 4930 organism/antibiotic combinations from 590 patients were evaluated. With regard to Gram-positive cocci (n=532), agreement between both methods was found in 93.9 % of cases, with 1.6 % very major, 1.1 % major and 2.6 % minor errors. For Gram-negative rods (n=226), agreement was found in 91.9 % of cases, with 1.2 % very major, 0.7 % major and 6.3 % minor errors. When applying the breakpoints of the Deutsches Institut fur Normung for interpretation of MICRONAUT tests, agreement of direct disk diffusion with standard testing decreased to 82.4 % in Gram-negative rods, with 3.6 % very major, 0.5 % major and 13.4 % minor errors. A high rate of disagreement was observed with oxacillin and gentamicin in Gram-positive cocci, and with cefuroxime, amoxycillin/clavulanate and piperacillin/tazobactam in Gram-negative rods. In conclusion, the limitations of direct disk diffusion testing of positive blood cultures must be kept in mind by the clinical microbiologist and should, where necessary, be communicated to the clinician to ensure adequate treatment of severely ill patients.
Andreas Edelmann - One of the best experts on this subject based on the ideXlab platform.
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Comparison of direct disk diffusion and standard microtitre Broth Dilution susceptibility testing of blood culture isolates.
Journal of medical microbiology, 2020Co-Authors: Andreas Edelmann, Tim Pietzcker, Nele WellinghausenAbstract:Bloodstream infections are life-threatening conditions which require timely initiation of appropriate antimicrobial therapy. The accuracy of direct disk diffusion susceptibility testing of positive blood cultures was investigated, including for the first time beta-lactam/beta-lactam-inhibitor combination antibiotics. Results of direct testing, following the guidelines of the Clinical and Laboratory Standards Institute, were compared to standard microtitre Broth Dilution susceptibility testing of the subcultured isolate on the Merlin MICRONAUT system. Altogether, 758 isolates and 4930 organism/antibiotic combinations from 590 patients were evaluated. With regard to Gram-positive cocci (n=532), agreement between both methods was found in 93.9% of cases, with 1.6% very major, 1.1% major and 2.6% minor errors. For Gram-negative rods (n=226), agreement was found in 91.9% of cases, with 1.2% very major, 0.7% major and 6.3% minor errors. When applying the breakpoints of the Deutsches Institut für Normung for interpretation of MICRONAUT tests, agreement of direct disk diffusion with standard testing decreased to 82.4% in Gram-negative rods, with 3.6% very major, 0.5% major and 13.4% minor errors. A high rate of disagreement was observed with oxacillin and gentamicin in Gram-positive cocci, and with cefuroxime, amoxycillin/clavulanate and piperacillin/tazobactam in Gram-negative rods. In conclusion, the limitations of direct disk diffusion testing of positive blood cultures must be kept in mind by the clinical microbiologist and should, where necessary, be communicated to the clinician to ensure adequate treatment of severely ill patients.
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comparison of direct disk diffusion and standard microtitre Broth Dilution susceptibility testing of blood culture isolates
Journal of Medical Microbiology, 2007Co-Authors: Andreas Edelmann, Tim Pietzcker, Nele WellinghausenAbstract:Bloodstream infections are life-threatening conditions which require timely initiation of appropriate antimicrobial therapy. The accuracy of direct disk diffusion susceptibility testing of positive blood cultures was investigated, including for the first time β-lactam/β-lactam-inhibitor combination antibiotics. Results of direct testing, following the guidelines of the Clinical and Laboratory Standards Institute, were compared to standard microtitre Broth Dilution susceptibility testing of the subcultured isolate on the Merlin MICRONAUT system. Altogether, 758 isolates and 4930 organism/antibiotic combinations from 590 patients were evaluated. With regard to Gram-positive cocci (n=532), agreement between both methods was found in 93.9 % of cases, with 1.6 % very major, 1.1 % major and 2.6 % minor errors. For Gram-negative rods (n=226), agreement was found in 91.9 % of cases, with 1.2 % very major, 0.7 % major and 6.3 % minor errors. When applying the breakpoints of the Deutsches Institut fur Normung for interpretation of MICRONAUT tests, agreement of direct disk diffusion with standard testing decreased to 82.4 % in Gram-negative rods, with 3.6 % very major, 0.5 % major and 13.4 % minor errors. A high rate of disagreement was observed with oxacillin and gentamicin in Gram-positive cocci, and with cefuroxime, amoxycillin/clavulanate and piperacillin/tazobactam in Gram-negative rods. In conclusion, the limitations of direct disk diffusion testing of positive blood cultures must be kept in mind by the clinical microbiologist and should, where necessary, be communicated to the clinician to ensure adequate treatment of severely ill patients.
William W Hope - One of the best experts on this subject based on the ideXlab platform.
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eucast technical note on the eucast definitive document edef 7 2 method for the determination of Broth Dilution minimum inhibitory concentrations of antifungal agents for yeasts edef 7 2 eucast afst
Clinical Microbiology and Infection, 2012Co-Authors: M C Arendrup, Manuel Cuencaestrella, Cornelia Lassflorl, William W HopeAbstract:Abstract The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has revised the EDef 7.1 document on the method for the determination of Broth Dilution minimum inhibitory concentrations of antifungal agents for fermentative yeasts. Changes are: dimethylsulphoxide is now the recommended solvent for caspofungin, micafungin and fluconazole; the shelf-life of plates containing the echinocandins prepared from stock solutions in dimethylsulphoxide is extended to 6 months at −80°C; testing of amphotericin and Cryptococcus has been incorporated; and minimum inhibitory concentration ranges for quality control strains and anidulafungin are included.
Yukio Osada - One of the best experts on this subject based on the ideXlab platform.
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antimicrobial susceptibilities of mycoplasma genitalium strains examined by Broth Dilution and quantitative pcr
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: Ryoichi Hamasuna, Jørgen Skov Jensen, Yukio OsadaAbstract:Mycoplasma genitalium is one of the important pathogens in male nongonococcal urethritis. However, the optimal treatment for the infection has not been clearly established. In vitro antimicrobial susceptibility data are also limited because of the difficulties in isolating M. genitalium strains from clinical specimens. In recent clinical trials, azithromycin (AZM) and moxifloxacin (MXF) effectively eradicated M. genitalium and improved symptoms (2, 11). We isolated M. genitalium strains from clinical specimens by cocultivation with Vero cells (4, 5, 9, 10), and 15 strains were adapted to SP4 mycoplasma medium. In the present study, antimicrobial susceptibility testing was performed by a conventional Broth Dilution method for these clinical isolates and for ATCC reference strains. Seven ATCC strains (G37, M30, R32, TW10-5G, TW10-6G, TW48-5G, and UTMB-10G) and an early passage of the M30 strain were obtained from the Mycoplasma Laboratory, Statens Serum Institut, Copenhagen, Denmark. The remaining strains were primary isolates obtained from male urethral or urine sediment specimens from the same laboratory. M2282, M2300, M2321, and M2341 were from Danish men. M6257, M6280, M6285, M6286, and M6328 were from Swedish men. M6090 and M6151 were from two consecutive specimens from a French man. M6282, M6283, M6284, and M6287 were from Japanese men. All strains were regrown in modified SP4 mycoplasma medium in the Laboratory of Microbiology, Frontier Science Research Center, University of Miyazaki, Japan. The modified SP4 mycoplasma medium was made in-house according to Tully's recipe (13) but without any antimicrobials. The pH of the medium was adjusted to 7.5. Antimicrobials for susceptibility testing were sitafloxacin (STX), MXF, levofloxacin (LVX), gatifloxacin (GAT), ciprofloxacin (CIP), norfloxacin (NOR), minocycline (MIN), doxycycline (DOX), tetracycline (TET), clarithromycin (CLR), and AZM. STX and LVX were supplied by Daiichi-Sankyo Co. Ltd., Tokyo, Japan. GAT was supplied by Kyorin Pharmaceutical Co. Ltd., Tokyo, Japan. CLR was supplied by Taisho Pharmaceutical Co., Ltd., Tokyo, Japan. AZM was supplied by Pfizer Global Research and Development, Pfizer Inc., Groton, CT. CIP and MXF were supplied by Bayer Health Care A.G., Leverkusen, Germany. NOR, MIN, DOX, and TET were bought from Sigma-Aldrich Inc., St. Louis, MO. The antimicrobial susceptibility tests (4) were performed by the modified Broth Dilution method as described by Hannan (6). In brief, antimicrobials were twofold diluted in SP4 mycoplasma medium. The thawed M. genitalium strains were diluted with SP4 mycoplasma medium to contain 104 color changing units/0.1 ml. A 0.025-ml volume of the Dilution of the antimicrobial was mixed with 0.075 ml of the diluted M. genitalium strains in a Nunclon Delta 96-well microtiter plate (96 MicroWell plates; Nunc, Roskilde, Denmark). The microtiter plates were sealed and incubated at 37°C for up to 4 weeks. The MICs were determined when an initial color change in the control wells without antimicrobials was observed. The MIC50s, MIC90s, and ranges for the 23 strains as determined by the Broth Dilution method are shown in Table Table1.1. Macrolide MICs for M6257 were high (MICs of AZM and CLR by the Broth Dilution method were 250 and 128 mg/liter, respectively). The strain was isolated from a patient failing AZM treatment (9). We have previously presented the antimicrobial susceptibility data for 27 M. genitalium strains, determined by measuring growth inhibition of M. genitalium in Vero cell culture by quantitative TaqMan PCR (4, 9). This method was also used to report six macrolide-resistant M. genitalium strains (3, 9). The MICs for 18 M. genitalium strains excluding 5 ATCC strains, as determined by this method, are also shown in Table Table1.1. Some data are lacking because some of the strains adapted to SP4 mycoplasma medium did not grow well in the Vero cell culture. However, it is important to compare data obtained by the two methods in order to be able to compare with other published MICs (1). MICs of macrolides determined by quantitative TaqMan PCR were slightly higher than those determined by the Broth Dilution method. However, the MICs for macrolide-resistant M. genitalium strains of macrolides are extremely high, and the differences between the two methods have no clinical relevance. MICs of fluoroquinolones and TETs determined by the two methods were similar. TABLE 1. MICs of M. genitalium as determined by conventional Broth Dilution and real-time quantitative PCR methods Among the tested antimicrobials, AZM and CLR were the most potent except against macrolide-resistant strain M6257. Among fluoroquinolones, STX and MFX were the most active drugs. AZM should probably be considered the first line of treatment for M. genitalium infections. However, several uncontrolled trials suggest that <85% of M. genitalium-positive patients are cured by a single dose of 1 g AZM, and recent findings have shown that a high proportion of those failing this treatment may develop macrolide resistance (3, 9). Consequently, AZM should be given as a 5-day treatment with 500 mg on day 1 followed by 250 mg on days 2 to 5 (2, 8). If patients fail this treatment, MFX is at present the only drug presenting a satisfactory cure rate (11). These recommendations reflect the antimicrobial susceptibility data (4). CLR and STX also appear potent in vitro, but clinical trials with these antimicrobials have not been done. STX was developed by Daiichi-Sankyo Co. Ltd., Japan, and has high in vitro activity against many bacteria including mycoplasmas (12) but is at the moment not registered in Europe and the United States. GAT has activity intermediate between those of MFX and LVX. It was withdrawn from the market September 2008, but in a recent study (R. Hamasuna et al., unpublished data) it provided a good clinical and microbiological response in cases of male nongonococcal urethritis. In the present study, MICs of the ATCC strains are very close (Table (Table2);2); this is not surprising as these strains are genetically extremely homogeneous (7). The range of MICs varies between one- and twofold among all tested antimicrobials. For some strains, our data differ slightly from those presented by Bebear et al., which were based on the agar Dilution method (1). One explanation may be that the MICs determined by the Broth Dilution method depend on the inoculum (6). In the present study, the inocula of the tested strains were examined by quantitative TaqMan PCR and were 2 × 104 to 8 × 104 genome equivalents per milliliter. TABLE 2. Differentiation of MIC range of M. genitalium between ATCC strains and non-ATCC strains by the Broth Dilution method
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Antimicrobial Susceptibilities of Mycoplasma genitalium Strains Examined by Broth Dilution and Quantitative PCR
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: Ryoichi Hamasuna, Jørgen Skov Jensen, Yukio OsadaAbstract:Mycoplasma genitalium is one of the important pathogens in male nongonococcal urethritis. However, the optimal treatment for the infection has not been clearly established. In vitro antimicrobial susceptibility data are also limited because of the difficulties in isolating M. genitalium strains from clinical specimens. In recent clinical trials, azithromycin (AZM) and moxifloxacin (MXF) effectively eradicated M. genitalium and improved symptoms (2, 11). We isolated M. genitalium strains from clinical specimens by cocultivation with Vero cells (4, 5, 9, 10), and 15 strains were adapted to SP4 mycoplasma medium. In the present study, antimicrobial susceptibility testing was performed by a conventional Broth Dilution method for these clinical isolates and for ATCC reference strains. Seven ATCC strains (G37, M30, R32, TW10-5G, TW10-6G, TW48-5G, and UTMB-10G) and an early passage of the M30 strain were obtained from the Mycoplasma Laboratory, Statens Serum Institut, Copenhagen, Denmark. The remaining strains were primary isolates obtained from male urethral or urine sediment specimens from the same laboratory. M2282, M2300, M2321, and M2341 were from Danish men. M6257, M6280, M6285, M6286, and M6328 were from Swedish men. M6090 and M6151 were from two consecutive specimens from a French man. M6282, M6283, M6284, and M6287 were from Japanese men. All strains were regrown in modified SP4 mycoplasma medium in the Laboratory of Microbiology, Frontier Science Research Center, University of Miyazaki, Japan. The modified SP4 mycoplasma medium was made in-house according to Tully's recipe (13) but without any antimicrobials. The pH of the medium was adjusted to 7.5. Antimicrobials for susceptibility testing were sitafloxacin (STX), MXF, levofloxacin (LVX), gatifloxacin (GAT), ciprofloxacin (CIP), norfloxacin (NOR), minocycline (MIN), doxycycline (DOX), tetracycline (TET), clarithromycin (CLR), and AZM. STX and LVX were supplied by Daiichi-Sankyo Co. Ltd., Tokyo, Japan. GAT was supplied by Kyorin Pharmaceutical Co. Ltd., Tokyo, Japan. CLR was supplied by Taisho Pharmaceutical Co., Ltd., Tokyo, Japan. AZM was supplied by Pfizer Global Research and Development, Pfizer Inc., Groton, CT. CIP and MXF were supplied by Bayer Health Care A.G., Leverkusen, Germany. NOR, MIN, DOX, and TET were bought from Sigma-Aldrich Inc., St. Louis, MO. The antimicrobial susceptibility tests (4) were performed by the modified Broth Dilution method as described by Hannan (6). In brief, antimicrobials were twofold diluted in SP4 mycoplasma medium. The thawed M. genitalium strains were diluted with SP4 mycoplasma medium to contain 104 color changing units/0.1 ml. A 0.025-ml volume of the Dilution of the antimicrobial was mixed with 0.075 ml of the diluted M. genitalium strains in a Nunclon Delta 96-well microtiter plate (96 MicroWell plates; Nunc, Roskilde, Denmark). The microtiter plates were sealed and incubated at 37°C for up to 4 weeks. The MICs were determined when an initial color change in the control wells without antimicrobials was observed. The MIC50s, MIC90s, and ranges for the 23 strains as determined by the Broth Dilution method are shown in Table Table1.1. Macrolide MICs for M6257 were high (MICs of AZM and CLR by the Broth Dilution method were 250 and 128 mg/liter, respectively). The strain was isolated from a patient failing AZM treatment (9). We have previously presented the antimicrobial susceptibility data for 27 M. genitalium strains, determined by measuring growth inhibition of M. genitalium in Vero cell culture by quantitative TaqMan PCR (4, 9). This method was also used to report six macrolide-resistant M. genitalium strains (3, 9). The MICs for 18 M. genitalium strains excluding 5 ATCC strains, as determined by this method, are also shown in Table Table1.1. Some data are lacking because some of the strains adapted to SP4 mycoplasma medium did not grow well in the Vero cell culture. However, it is important to compare data obtained by the two methods in order to be able to compare with other published MICs (1). MICs of macrolides determined by quantitative TaqMan PCR were slightly higher than those determined by the Broth Dilution method. However, the MICs for macrolide-resistant M. genitalium strains of macrolides are extremely high, and the differences between the two methods have no clinical relevance. MICs of fluoroquinolones and TETs determined by the two methods were similar. TABLE 1. MICs of M. genitalium as determined by conventional Broth Dilution and real-time quantitative PCR methods Among the tested antimicrobials, AZM and CLR were the most potent except against macrolide-resistant strain M6257. Among fluoroquinolones, STX and MFX were the most active drugs. AZM should probably be considered the first line of treatment for M. genitalium infections. However, several uncontrolled trials suggest that
Wendy Murro - One of the best experts on this subject based on the ideXlab platform.
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a rapid Broth Dilution technique utilizing hemoglobin for determining antibiotic susceptibility of microorganisms
Journal of Microbiological Methods, 1994Co-Authors: Marvin Murray, Wendy MurroAbstract:Abstract A 3–4 h antibiotic susceptibility method based on hemoglobin reduction was developed. The susceptibility of bacteria to antibiotics was determined by growing the bacteria in liquid media in the presence of antibiotics and partially purified hemoglobin. Oxyhemoglobin (541 and 576 nm) and reduced hemoglobin (556 nm) have distinct spectroscopic absorption bands. A decrease in oxygen consumption by the bacteria inhibited by the antibiotic was indicated by the hemoglobin remaining in the oxyhemoglobin form. Resistant bacteria continued to consume oxygen, forming reduced hemoglobin. A Perkin-Elmer Lambda 5 was used to scan the samples from 500–600 nm. The two oxyhemoglobin peaks indicate susceptibility and the one reduced peak indicates resistance. The minimum inhibitory concentration (the first antibiotic concentration that remained in the oxyhemoglobin form) obtained with this method corresponded with literature values and current minimum inhibitory concentration techniques.
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a rapid Broth Dilution method for recognizing methicillin resistant staphylococci
Journal of Microbiological Methods, 1993Co-Authors: Marvin Murray, Wendy MurroAbstract:Abstract Methicillin resistant Staphylococcus aureus is a clinical and epidemiological problem in hospitals. The difficulty in detecting and defining methicillin-resistant Staphylococci has been a problem in the past because of the difference in the growth characteristics of the organisms that make up the diverse population. Each population is made up of methicillin-susceptible and methicillin-resistant organisms which grow slower, prefer a higher salt content and lower temperatures. The susceptibility of the staphylococci to oxacillin can be determined in 3–4 h utilizing the oxygen consumption method. The bacteria were grown in MHB + 4% NaCl in varying concentrations of oxacillin (0.5–8 mg/l). Oxygen consumption was measured using an oxygen electrode in the samples containing antibiotic and compared to a positive and negative control. The bacteria that continued to consume the oxygen were not inhibited by the antibiotic. The MIC values can be determined from the oxygen consumption readings. Ninety two clinical isolates were tested and 96% demonstrated agreement with the reference methods used.