The Integrity of Heme Is Essential For Reproducible Detection of Metronidazole-Resistant Clostridioides difficile By Agar Dilution Susceptibility Tests

Metronidazole resistance in clinical Clostridioides difficile is often described as unstable, since resistant strains reportedly appear susceptible following freezer storage or brief passage. This has presented a conundrum for adopting susceptibility testing to accurately evaluate the connection between metronidazole resistance and decreased clinical efficacy of metronidazole in patients with C. difficile infections (CDIs). We discovered that supplementation of microbiological media with the metalloporphyrin heme is crucial for detection of metronidazole-resistant C. difficile using the agar dilution susceptibility testing method. Known metronidazole-resistant strains appeared susceptible to metronidazole in media lacking heme. Similarly, these resistant strains exhibited increased susceptibility to metronidazole when tested on heme-containing agars that were exposed to room light, for more than 1 day, likely due to heme photodecomposition. In parallel experiments, resistance was reproducibly detected when heme-containing agars were either prepared and used on the same day or were protected from light and then used on subsequent days. Notably, heme did not influence the susceptibilities of drug-susceptible strains that were of the same ribotype as the resistant strains. These findings firmly show that the consistent detection of metronidazole-resistant C. difficile is dependent upon heme and its protection from light. Studies are warranted to determine the extent to which this heme-associated metronidazole-resistant phenotype affects the clinical efficacy of metronidazole in CDI and the underlying genetic and biochemical mechanism.

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E-Test or Agar Dilution for Metronidazole Susceptibility Testing of Helicobacter Pylori: Importance of the Prevalence of Metronidazole Resistance

10.21203/rs.3.rs-677597/v1 ◽

2021 ◽

Author(s):

Jinnan Chen ◽

Yu Huang ◽

Zhaohui Ding ◽

Xiao Liang ◽

Hong Lu

Keyword(s):

Helicobacter Pylori ◽

Susceptibility Testing ◽

Resistance Rate ◽

Test Method ◽

Major Error ◽

Metronidazole Resistance ◽

Agar Dilution ◽

H Pylori ◽

High Level ◽

Level Resistance

Abstract Background: A number of studies have shown that E-test overestimated the presence of Helicobacter pylori (H. pylori) resistance compared to agar dilution.Objective: The purpose of this study was to explore whether E-test could be an alternative for agar dilution to detect the metronidazole susceptibility of H. pylori.Method: E-test and agar dilution were used to assess susceptibility of H. pylori to metronidazole, clarithromycin and levofloxacin in 281 clinical isolates obtained from China where resistance was high. Cohen kappa analysis, McNemar test, essential and categorical agreement analysis were performed for these two methods. Results: Overall, the result of E-test showed similar prevalence of resistance rate to all antibiotics compared with agar dilution. The essential agreement (EA) of E-test method and agar dilution in the evaluation susceptibility of H. pylori to clarithromycin and levofloxacin were moderate, with 89.0% and 79.7% respectively, but only 45.9% for metronidazole. Results showed categorical agreement (CA) between E-test and agar dilution were 100% for both clarithromycin and levofloxacin. As for metronidazole, the CA was 98.7%, no major error was identified, and rate of very major error was 1.8%.Conclusion: E-test can be an alternative method to detect the metronidazole susceptibility of H. pylori in regions where high-level resistance is common.

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Variation in erythromycin and clindamycin susceptibilities of Streptococcus pneumoniae by four test methods.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.1.129 ◽

1997 ◽

Vol 41 (1) ◽

pp. 129-134 ◽

Cited By ~ 39

Author(s):

E L Fasola ◽

S Bajaksouzian ◽

P C Appelbaum ◽

M R Jacobs

Keyword(s):

Streptococcus Pneumoniae ◽

Susceptibility Testing ◽

Ambient Air ◽

Test Methods ◽

Disk Diffusion ◽

Broth Microdilution ◽

Microdilution Method ◽

Broth Microdilution Method ◽

Agar Dilution ◽

Resistant Strains

Susceptibilities of 124 strains of Streptococcus pneumoniae to erythromycin and clindamycin were determined by the National Committee for the Clinical Laboratory Standards (NCCLS) broth microdilution method, with incubation for 20 to 24 h in ambient air and with modifications of this method by incubation for up to 48 h in air and CO2. Strains were also tested by agar dilution, E-test, and disk diffusion; good correlation was obtained with these methods, with clear separation into bimodal populations of susceptible and resistant stains. The broth microdilution method, however, using incubation in air for 24 h (NCCLS method), misclassified 4 of 92 erythromycin-resistant strains (1 as susceptible and 3 as intermediate) and 25 of 58 clindamycin-resistant strains (all as susceptible). With the exception of one strain with clindamycin, susceptible and resistant strains were correctly classified by the microdilution method with incubation in CO2 for 24 h or in ambient air for 48 h. Disk diffusion, agar dilution, and E-test methods with incubation in 5% CO2 are therefore reliable methods for susceptibility testing of pneumococci against these agents. However, the NCCLS microdilution method, which specifies incubation for 20 to 24 h in ambient air, produced significant very major errors (43%) clindamycin. Modification of the microdilution method by incubation in 5% CO2 or by extension of incubation time in ambient air to 48 h corrected these errors. Disk diffusion, however, was shown to be a simple, convenient, and reliable method for susceptibility testing of pneumococci to erythromycin and clindamycin and is suggested as the method of choice for these agents.

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In VitroActivity of Fosfomycin against a Collection of Clinical Pseudomonas aeruginosa Isolates from 16 Spanish Hospitals: Establishing the Validity of Standard Broth Microdilution as Susceptibility Testing Method

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00589-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5701-5703 ◽

Cited By ~ 21

Author(s):

María Díez-Aguilar ◽

María-Isabel Morosini ◽

Rosa del Campo ◽

María García-Castillo ◽

Javier Zamora ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Susceptibility Testing ◽

Disk Diffusion ◽

Dilution Method ◽

Agar Dilution Method ◽

Broth Microdilution ◽

Content Type ◽

Testing Method ◽

Broth Microdilution Method ◽

Agar Dilution

ABSTRACTThe broth microdilution method for fosfomycin andPseudomonas aeruginosawas assessed and compared with the approved agar dilution method in 206 genetically unrelatedP. aeruginosaclinical isolates. Essential agreement between the two methods was 84%, and categorical agreement was 89.3%. Additionally, Etest and disk diffusion assays were performed. Results validate broth microdilution as a reliable susceptibility testing method for fosfomycin againstP. aeruginosa. Conversely, unacceptable concordance was established between Etest and disk diffusion results with agar dilution results.

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Comparative Evaluation of Colistin Susceptibility Testing Methods among Carbapenem-Nonsusceptible Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00868-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4625-4630 ◽

Cited By ~ 57

Author(s):

Konstantina Dafopoulou ◽

Olympia Zarkotou ◽

Evangelia Dimitroulia ◽

Christos Hadjichristodoulou ◽

Vasiliki Gennimata ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Acinetobacter Baumannii ◽

Susceptibility Testing ◽

Reference Method ◽

Test Strip ◽

Clinical Isolates ◽

Content Type ◽

Gradient Diffusion ◽

Agar Dilution ◽

Resistant Strains

ABSTRACTWe compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptibleKlebsiella pneumoniae(n= 41) andAcinetobacter baumannii(n= 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 μg/ml, respectively, were applied for bothK. pneumoniaeandA. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST.

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Antibiotic Susceptibilities ofHelicobacter pyloriStrains Isolated in the Province of Alberta

Canadian Journal of Gastroenterology ◽

10.1155/1998/672746 ◽

1998 ◽

Vol 12 (4) ◽

pp. 295-298 ◽

Cited By ~ 12

Author(s):

Diane E Taylor ◽

Qin Jiang ◽

Richard N Fedorak

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Gastric Biopsy ◽

Minimal Inhibitory Concentrations ◽

Antibiotic Susceptibilities ◽

Biopsy Specimens ◽

Metronidazole Resistance ◽

Agar Dilution ◽

Resistant Strains ◽

H Pylori

The incidence of antibiotic resistance to amoxicillin, clarithromycin, erythromycin, metronidazole and tetracycline inHelicobacter pyloristrains isolated from gastric biopsy specimens obtained in Alberta was investigated. Results for all antibiotics were obtained using agar dilution, and in addition to metronidazole, the E test was used. Resistance to amoxicillin and tetracycline was not detected. Metronidazole resistance determined using agar dilution was approximately 12% (95% CI 4% to 26%) when minimal inhibitory concentrations (MICs) were at least 8 µg/mL, but fell to 2% (95% CI 0.1% to 13%) when MICs were set at 32 µg/mL or greater. The E test for metronidazole resistance (MIC 8 µg/mL or greater) yielded a slightly higher percentage of resistant strains compared with agar dilution tests (14%, 95% CI 5% to 29%). One of the 31 strains was resistant to clarithromycin (MIC 8 µg/mL) and erythromycin (MIC 16 µg/mL). Thus, the incidence of resistance to clarithromycin, part of the currently used triple therapy for eradication ofH pylori, was 3% (95% CI 0.1% to 17%).

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2166. Performance Study on the New ETEST® Piperacillin/Tazobactam (P/T) MIC Strip

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.1846 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S735-S735

Author(s):

Roland Martelin ◽

Edwige Pillon ◽

Emilie Cutivet ◽

Marion Pompilio ◽

Diane Halimi ◽

...

Keyword(s):

Susceptibility Testing ◽

Inhibitory Concentration ◽

Reference Method ◽

Error Rates ◽

Performance Study ◽

First Line ◽

Severe Infections ◽

Agar Dilution ◽

Resistant Strains ◽

High Level

Abstract Background Piperacillin/Tazobactam combination is a first-line antibiotic and carbapenem sparing option for severe infections due to Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. ETEST® strips allow to determine antimicrobial Minimum Inhibitory Concentration (MIC). ETEST® Piperacillin/Tazobactam (Ptc) was developped in 1995 against agar dilution reference method. Since then, resistance to Piperacillin/Tazobactam has been increasing and broth microdilution (BMD) substituted for agar dilution as the reference method. The new ETEST® P/T strip for determining MIC of Enterobacteriaceae, P. aeruginosa and A. baumannii was developed against BMD using a panel of recent strains well genotypically characterized. The aim of this study was to compare the performance of both strips on a panel of challenging strains harboring different-resistant mechanisms. Methods A total of 64 strains were tested using ETEST® P/T, ETEST® PTc and BMD: 48 Enterobacteriaceae including 25 resistant strains and 16 P. aeruginosa including 11 resistant strains. The results were analyzed for essential (EA) and category (CA) agreements, minor (mE), major (ME) and very major (VME) error rates using FDA/CLSI 2019 breakpoints (Enterobacteriaceae, P. aeruginosa: ≤ 16/4(S); ≥ 128/4(R) µg/mL). Results Although the panel of strains was challenging including different resistant mechanisms (acquired penicillinase, high-level cephalosporinase, acquired cephalosporinase, ESBL, carbapenemase), the new ETEST® P/T performance was significantly improved for Enterobacteriaceae with an EA at 92,2% without ME or VME. This improvement was also linked to the easiest reading (significant decrease of microcolonies in the ellipse zone). For P. aeruginosa, the performance was similar between the two strips but the new ETEST® P/T was better correlated with the BMD and showed an EA of 100%. The results are summarized in the table. Conclusion The new ETEST® P/T improved the MIC determination and resistance detection, as well as the reading of MIC end points for the routine use. This study emphasizes the need to check the performance of the antimicrobial susceptibility testing products by testing strains reflecting the current epidemiology. Disclosures All authors: No reported disclosures.

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Antimicrobial Susceptibility Testing ofHelicobacter pylori in a Large Multicenter Trial: the MACH 2 Study

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.11.2747 ◽

1999 ◽

Vol 43 (11) ◽

pp. 2747-2752 ◽

Cited By ~ 162

Author(s):

Francis Mégraud ◽

Norbert Lehn ◽

Tore Lind ◽

Ekkehard Bayerdörffer ◽

Colm O’Morain ◽

...

Keyword(s):

Susceptibility Testing ◽

Point Mutations ◽

Multicenter Trial ◽

Dilution Method ◽

Agar Dilution Method ◽

Secondary Resistance ◽

Agar Dilution ◽

Resistant Strains ◽

H Pylori ◽

The Impact

ABSTRACT Culture and susceptibility testing of Helicobacter pylori strains was performed in a large multinational, multicenter randomized clinical trial. Culture was carried out on gastric biopsy samples obtained from 516 patients at entry and had a sensitivity of 99% when the [13C]urea breath test was used as a reference. Susceptibility testing was performed for clarithromycin and metronidazole on 485 strains by an agar dilution method and the epsilometer test (Etest) and for amoxicillin by an agar dilution method only. Resistance to clarithromycin (>1 μg/ml) was found in 3% of the H. pylori strains, with a perfect correlation between Etest and agar dilution methods. Resistance to metronidazole (>8 μl/ml) was found in 27% of the strains by agar dilution, but there were important discrepancies between it and the Etest method. No resistance to amoxicillin was found. The logarithms of the MICs of the three antibiotics against susceptible strains had a distribution close to normal. The impact of resistance was tested in the four arms of the trial. There were not enough clarithromycin-resistant strains to evaluate the impact of resistance on the cure rate of clarithromycin-based regimens. For metronidazole-resistant strains, the impact noted in the clarithromycin-metronidazole arm was partially overcome when omeprazole was added (76% eradication for resistant strains versus 95% for susceptible strains). Secondary resistance to clarithromycin occurred in strains from 12 of 105 patients (11.4%) after the failure of a clarithromycin-based regimen to effect eradication. The detection of point mutations in clarithromycin-resistant strains was performed by a combination of PCR and restriction fragment length polymorphism. Mutations (A2142G and 2143G) were found in all strains tested except one. This study stresses the importance of performing susceptibility tests in clinical trials in order to explain the results of different treatments.

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Phenotypic and Molecular Characterization of Mycobacterium tuberculosis Isolates Resistant to both Isoniazid and Ethambutol

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2218-2225.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2218-2225 ◽

Cited By ~ 53

Author(s):

Linda M. Parsons ◽

Max Salfinger ◽

Anne Clobridge ◽

Jillian Dormandy ◽

Lisa Mirabello ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Susceptibility Testing ◽

Rapid Screening ◽

The Past ◽

Resistant Strains ◽

The Common ◽

Susceptibility Tests ◽

High Level ◽

Level Resistance

ABSTRACT In performing radiometric susceptibility testing on over 2,000 patient isolates of Mycobacterium tuberculosis during the past 6 years, we found that resistance to 7.5 μg/ml ethambutol (EMB) occurred only in isolates that are also resistant to 0.4 μg/ml isoniazid (INH). Using 157 selected isolates in the present study, we performed radiometric and agar proportion susceptibility tests and DNA sequencing of genetic regions associated with resistance to these two drugs. The goal was to study the occurrence of the common mutations associated with resistance to each drug and also to determine whether any particular INH-resistance-associated mutation occurred more often in combination with any particular EMB-resistance-associated mutation. In an analysis of 128 isolates resistant to 0.4 μg/ml INH, we found that a mutation at katG Ser315 was more common in isolates also resistant to 7.5 μg/ml EMB (61 of 67 = 91.0%) than in isolates either susceptible to EMB or resistant to 2.5 μg/ml EMB (39 of 60 = 65.0%). These observations suggest that INH-resistant strains with a mutation at katG Ser315 are more likely to acquire resistance to 7.5 μg/ml EMB than are isolates with INH-resistance-associated mutations at other sites. In addition, we found that 64 of 67 (95.5%) isolates resistant to 7.5 μg/ml EMB contained a mutation in either codon 306 or codon 406 of embB. Met306Val was the most common embB mutation, present in 52 (77.6%) of the 67 isolates. Most occurrences of this mutation (49 of 52 = 94.2%) were found in isolates that also contained the katG Ser315Thr mutation. Finally, sequencing this region of embB appears to be sufficiently sensitive for use as a rapid screening tool for detection of high-level resistance to EMB.

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Echinocandin Susceptibility Testing of Candida Species: Comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest, Disk Diffusion, and Agar Dilution Methods with RPMI and IsoSensitest Media

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01256-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 426-439 ◽

Cited By ~ 112

Author(s):

Maiken Cavling Arendrup ◽

Guillermo Garcia-Effron ◽

Cornelia Lass-Flörl ◽

Alicia Gomez Lopez ◽

Juan-Luis Rodriguez-Tudela ◽

...

Keyword(s):

Susceptibility Testing ◽

Hot Spot ◽

Disk Diffusion ◽

Wild Type ◽

Limit Value ◽

Test Parameters ◽

Agar Dilution ◽

Susceptibility Tests ◽

Distribution Distance ◽

Two Populations

ABSTRACT This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC50) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S ≤ 2 μg/ml) for CLSI results, 89.2% fks hot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fks hot spot mutants.

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Comparative analysis of novel suspension testing and agar cup diffusion methods in establishing the susceptibility of Pseudomonas aeruginosa against ethanol and chlorhexidine gluconate

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v11ispl2.2693 ◽

2020 ◽

Vol 11 (SPL2) ◽

pp. 271-275

Author(s):

Hemalatha E ◽

Bhuvaneshwari G ◽

Kalyani M

Keyword(s):

Pseudomonas Aeruginosa ◽

Comparative Analysis ◽

Susceptibility Testing ◽

Chlorhexidine Gluconate ◽

Diffusion Method ◽

Time Consumption ◽

Testing Method ◽

Resistant Strains ◽

Sensitive Method

Pseudomonas aeruginosa is one among the leading nosocomial pathogens worldwide. It is therefore necessary to decrease and to prevent a rebound of growth. Comparison of novel suspension testing method and agar cup diffusion method results in determination of the sensitive method to identify effectiveness of disinfectants against microbial activity. This study was carried out to determine the effectiveness among novel suspension testing and agar cup diffusion method to determine disinfectant susceptibility and also to identify the efficacy of ethanol and chlorhexidine gluconate at manufacturer’s concentration against Pseudomonas aeruginosa. In this study 50 isolates of Pseudomonas aeruginosa were included. Each isolate was subjected to novel suspension testing method and agar cup diffusion method with ethanol and chlorhexidine gluconate, the results were observed and recorded. The 50 isolates, sensitive strains showed 100% sensitivity to chlorhexidine gluconate and 95% to ethanol. Whereas resistant strains showed 100% sensitivity to chlorhexidine gluconate, 75% were sensitive to ethanol. Both agar cup diffusion method and novel suspension method yielded similar results. With the advantage of easy processing and less time consumption, agar cup diffusion method can be routinely used for determining the disinfectant susceptibility testing.

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