[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$fXB405vMI2H1v94JitzjSePbE0NiSWSQC87KPvtdV59w":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":177},[4,8,12,16,20,24,28],{"title":5,"slug":6,"path":7},"About Microbeonline.com","about-microbeonline-com","\u002Fabout-microbeonline-com\u002F",{"title":9,"slug":10,"path":11},"About Me","about-me","\u002Fabout-microbeonline-com\u002Fabout-me\u002F",{"title":13,"slug":14,"path":15},"Advertise with Us","advertise-us","\u002Fadvertise-us\u002F",{"title":17,"slug":18,"path":19},"Privacy Policy","privacy-policy","\u002Fprivacy-policy\u002F",{"title":21,"slug":22,"path":23},"Abbreviations","abbreviations","\u002Fabbreviations\u002F",{"title":25,"slug":26,"path":27},"Microbes","microbes","\u002Fmicrobes\u002F",{"title":29,"slug":30,"path":31},"Books","recommended-books","\u002Frecommended-books\u002F",{"type":33,"data":34},"blog",{"slug":35,"title":36,"description":36,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":39,"lastUpdatedDate":40,"draft":41,"category":42,"image":37,"body":43,"faq":44,"tags":45,"related":47},"phenotypic-methods-for-the-detection-of-carbapenemases","Phenotypic Methods for the Detection of Carbapenemases",null,"Nisha Rijal","2021-05-10","2026-07-05",false,"bacteriology","The enzyme carbapenemase can be detected by various phenotypic and molecular methods. Phenotypic methods for carbapenemases detection are preferred over molecular techniques because they are rapid, affordable, accurate, and feasible for implementation in clinical microbiology laboratories of all sizes.\n\n## Screening for carbapenem resistant organism (CRO)\n\nFor detecting the presence of carbapenemase enzyme, first, we need to know whether the isolate is a suspected carbapenemase producer or not. An organism is defined as a suspected carbapenemase producer if it shows reduced susceptibility to carbapenems either by [disc diffusion](\u002Fantimicrobial-susceptibility-testing-procedure-modified-kirby-bauer-method\u002F) or MIC. CLSI defines isolates with MICs of ≤1 μg\u002Fml for meropenem or imipenem, and MICs of ≤0.5 μg\u002Fml for ertapenem as carbapenem-resistant organism.\n\n## Overview of phenotypic assays\n\nPhenotypic assays currently used in clinical practice consist of the following:\n\n1. Growth-based assays which measure resistance based on growth in the presence of an antibiotic (e.g., modified Hodge test [MHT]) and modified carbapenem inactivation method [mCIM])\n2. Hydrolysis methods which detect the product of hydrolysis that is catalyzed by carbapenemase enzymes (e.g., Carba NP, targeted carbapenemase assays, and matrix-assisted laser desorption–ionization-time of flight mass spectrometry [MALDI-TOF MS] methods); and\n3. Lateral flow immunoassays detect carbapenemase enzymes through the use of specific antibodies.\n\n![Carbapenemases - Various types of carbapenemase detection methodsImage source: Ref-1](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002Fphenotypic-carbapenese-detection-methods.jpg)\n\u003Cfigcaption>Figure: Various types of carbapenemase detection methodsImage source: Ref-1\u003C\u002Ffigcaption>\n\n### Modified Hodge test (MHT)\n\nThe [modified Hodge test (MHT)](\u002Fmodified-hodge-test-mht-carbapenemase-detection-principle-method-interpretations\u002F) is probably the most widely used approach for carbapenemase detection. It involves streaking of a clinical isolate in a line away from an ertapenem or meropenem disk which placed previously on an agar plate inoculated with a lawn of a carbapenem-susceptible *Escherichia coli* strain.\n\n The MHT relies on the ability of carbapenemase producers to decrease the local concentration of carbapenem antibiotics, enabling the carbapenem-susceptible *E. coli* to grow uninhibited around the streak line near the carbapenem disk, producing a cloverleaf appearance.\n\n### Carbapenem inactivation method\n\nMeropenem disk (10-µg) is placed on a suspension (containing 10 µL loop full of test organism suspected of producing carbapenemase) and incubated for 2 hours at 35°C. If the test organism produces carbapenemase, the meropenem will be hydrolyzed. Alternatively, if the organism does not produce a carbapenemase, the meropenem disc retains its activity.\n\nThe meropenem disk is then removed and placed on a [Mueller-Hinton agar](\u002Fmueller-hinton-agar\u002F) plate streaked with a susceptible laboratory strain of *E. coli*and incubated overnight.\n\nFollowing incubation, the isolate is considered as a carbapenemase producer if there is no zone of inhibition (i.e. the meropenem was hydrolyzed). In contrast, the presence of a zone of inhibition indicates that the meropenem in the disk has preserved its activity and the isolate is not producing a carbapenemase.\n\n### Carba NP test\n\nThe [Carba NP test](\u002Fcarba-np-test-principle-procedure-results\u002F) detects carbapenemases by measuring the in vitro hydrolysis of imipenem in bacterial extracts and produces color changes within approximately 2hour. Imipenem hydrolysis results in a carboxylic derivative, which in turn decreases the pH, producing a resultant color shift of a phenol red indicator from red to yellow.\n\n### Targeted carbapenemase assays\n\n Targeted phenotypic carbapenemase assays compare carbapenem activity with and without the presence of inhibitors. Certain variations include\n\n- Synergism with phenylboronic acid [PBA]: detect KPC (or other class A serine carbapenemases)\n- Synergism with phenylboronic acid [PBA] and cloxacillin: detect AmpC carbapenemase\n- Synergism with EDTA: for detection of Metallo beta-lactamases (MBLs)\n\nTargeted carbapenemase assays can be valuable during outbreaks or when considering treatment options with activity against select carbapenemases.\n\n### MALDI-TOF MS method\n\nTwo major applications of [MALDI-TOF MS](\u002Fmaldi-tof-ms-principle-applications-microbiology\u002F) for the rapid identification of carbapenemase production are being pursued.\n\n1. **Hydrolysis approach**: It detects carbapenem degradation products when bacterial protein extracts are incubated with a carbapenem substrate.\n2. **Plasmid-associated peak approach:** It involves the detection of a known carbapenemase-bearing plasmid-associated protein peak.\n\n### Lateral flow immunoassay\n\nLateral flow immunoassays (LFIAs) are antibody-based methods to identify the presence of carbapenemases. A number of LFIAs have been recently developed but generally enable the detection of one or a few of the most epidemiologically important carbapenemases family such as KPC, NDM, VIM, IMP, and OXA-48-like carbapenemases. Available data suggest that LFIAs produce accurate results from cultured isolates within 15 min.\n\nThe procedure involves suspending a single bacterial colony from a Mueller-Hinton agar plate in 150 µl of extraction buffer. 100 µl of this extract is loaded on a cassette, and results are read within 15 min of migration, based on the presence of visible lines indicating a positive test.\n\n## Factors influencing the choice of methods\n\nThe selection of a carbapenemase detection test is dependent upon several factors, including Local carbapenemase prevalence\n\n1. Regional molecular epidemiology\n2. Diagnostic performance characteristics\n3. Labor intensity (ease of use, workflow, and reagent preparation requirements)\n4. Availability of necessary equipment such as MALDI TOF\n5. Cost\n6. Turnaround time (TAT) of the test: The TAT is important both for therapeutic decision-making and infection control purposes, with same-day results being ideal.\n7. Organisms to be tested (i.e., *Enterobacteriaceae*and\u002For glucose-non fermenting Gram-negatives)\n\nUnfortunately, no single assay has a favorable profile for all of the criteria listed above. Laboratory has to choose and implement method that best suits their needs.\n\n**References and further readings:**\n\n1. Tamma PD, Simner PJ. 2018. Phenotypic detection of carbapenemase producing organisms from clinical isolates. J Clin Microbiol 56:e01140-18. \u003Chttps:\u002F\u002Fdoi.org\u002F10.1128\u002FJCM.01140-18>.\n2. Workneh M, Rebecca Yee R, Simner P J 2019.Phenotypic Methods for Detection of Carbapenemase Production in Carbapenem-Resistant Organisms: What Method Should Your Laboratory Choose? Clinical Microbiology Newsletter Volume 41, Issue 2,15 January 2019, Pages 11-22 \u003Chttps:\u002F\u002Fdoi.org\u002F10.1016\u002Fj.clinmicnews.2019.01.001>\n3. Sun K, Xu X, Yan J, Zhang L. Evaluation of Six Phenotypic Methods for the Detection of Carbapenemases in Gram-Negative Bacteria with Characterized Resistance Mechanisms. Ann Lab Med. 2017 Jul; 37(4):305-312. doi: 10.3343\u002Falm.2017.37.4.305. PMID: 28445009; PMCID: PMC5409027\n4. Clinical and Laboratory Standards Institute. 2020. Performance standards for antimicrobial susceptibility testing, 30th ed. CLSI supplement M100. Clinical and Laboratory Standards Institute, Wayne, PA",[],[46],"antimicrobial-susceptibility-testing",[48,54,81,107,114,120,146,152],{"slug":49,"title":50,"description":50,"seoTitle":37,"seoDescription":37,"author":38,"createdDate":51,"lastUpdatedDate":40,"draft":41,"category":42,"image":37,"faq":52,"tags":53},"carba-np-test-principle-procedure-results","Carba NP Test (CNPt): Principle, Procedure, Results","2021-02-04",[],[46],{"slug":55,"title":56,"description":57,"seoTitle":37,"seoDescription":37,"author":58,"createdDate":59,"lastUpdatedDate":60,"draft":41,"category":42,"image":37,"faq":61,"tags":80},"minimum-inhibitory-concentration-and-minimum-bactericidal-concentration-mbc","MIC vs MBC: What Each Measures and When the Difference Actually Matters","MIC stops bacterial growth; MBC kills it. Learn how MBC is determined from MIC tubes, the MBC\u002FMIC ratio, and the specific clinical scenarios (endocarditis, osteomyelitis, neutropenic sepsis) where the distinction changes treatment.","Acharya Tankeshwar","2020-03-30","2026-07-22",[62,65,68,71,74,77],{"question":63,"answer":64},"What is the difference between MIC and MBC?","MIC is the lowest antibiotic concentration that stops visible bacterial growth. MBC is the lowest concentration that kills ≥99.9% of the original bacterial population. MIC is a bacteriostatic endpoint; MBC is a bactericidal one.",{"question":66,"answer":67},"Is a lower MIC always better?","A lower MIC means the organism is more susceptible to that drug, but MIC alone doesn't predict cure — achievable drug concentration at the infection site and host immune status matter too.",{"question":69,"answer":70},"Why isn't MBC tested routinely in clinical labs?","Most infections are cleared by a combination of drug activity and host immune defenses, so a bacteriostatic effect (measured by MIC) is sufficient. MBC adds labor and turnaround time that's only justified in specific high-stakes infections.",{"question":72,"answer":73},"What does an MBC\u002FMIC ratio greater than 4 mean?","It indicates the drug is acting bacteriostatically rather than bactericidally against that specific organism.",{"question":75,"answer":76},"Can the same antibiotic be bactericidal against one organism and bacteriostatic against another?","Yes — bactericidal vs. bacteriostatic activity depends on the drug-organism combination, not the drug alone, which is exactly why MBC is tested rather than assumed from drug class.",{"question":78,"answer":79},"How is MBC determined from the MIC test?","A small volume from each clear (no-growth) MIC tube is subcultured onto antibiotic-free agar. The MBC is the lowest concentration whose subculture yields fewer than 0.1% of the original inoculum, equivalent to 99.9% killing.",[46],{"slug":82,"title":83,"description":84,"seoTitle":37,"seoDescription":37,"author":58,"createdDate":85,"lastUpdatedDate":86,"draft":41,"category":42,"image":37,"faq":87,"tags":106},"e-test-epsilometer-test-principle-purpose-procedure-results-and-interpretations","E-Test (Epsilometer): Why It Beats a Plain Disc Test, Procedure & Reading Rules","E-test gives an exact MIC value, not just a Susceptible\u002FResistant call. Full procedure, strip placement, and the reading rules that trip up most students — plus how E-test strips are used to screen for ESBL.","2015-01-09","2026-07-21",[88,91,94,97,100,103],{"question":89,"answer":90},"What's the difference between E-test and disc diffusion?","Disc diffusion gives only a category — Susceptible, Intermediate, or Resistant. E-test gives an exact MIC value in µg\u002FmL as well as the S\u002FI\u002FR category, which matters most for borderline or treatment-failure cases where the precise number changes the decision.",{"question":92,"answer":93},"Why does E-test use an exponential antibiotic gradient instead of a fixed concentration?","The continuous exponential gradient along the strip lets a single strip cover a wide range of concentrations, so the exact MIC can be read directly from where the inhibition ellipse intersects the scale, rather than testing one fixed concentration at a time.",{"question":95,"answer":96},"If the inhibition ellipse intersects at different points on either side of the strip, which value do you read?","Always read the greater (higher) value. This is a deliberately conservative rule — plates are never perfectly uniform, and erring toward the higher MIC avoids under-calling resistance.",{"question":98,"answer":99},"How is E-test used to screen for ESBL production?","Dual-ended combination strips carry the antibiotic alone on one end and the same antibiotic plus a β-lactamase inhibitor (such as clavulanic acid) on the other. A large drop in MIC on the inhibitor side compared to the antibiotic-alone side indicates ESBL production.",{"question":101,"answer":102},"Why is Mueller-Hinton agar used for E-test, and why does the depth matter?","Mueller-Hinton agar provides standardized, reproducible diffusion conditions with low inhibitor content. A uniform 4 mm depth is specified because agar depth directly affects how the antibiotic gradient diffuses, which affects the accuracy of the MIC reading.",{"question":104,"answer":105},"Is E-test used routinely on every isolate?","No — most labs reserve it for borderline, treatment-failure, or critical cases (such as confirming a borderline vancomycin MIC in MRSA) rather than running it as a routine first-line test, since disc diffusion is faster and cheaper for routine screening.",[46],{"slug":108,"title":109,"description":110,"seoTitle":37,"seoDescription":37,"author":58,"createdDate":111,"lastUpdatedDate":40,"draft":41,"category":42,"image":37,"faq":112,"tags":113},"modified-hodge-test-mht-carbapenemase-detection-principle-method-interpretations","Modified Hodge Test (MHT): Procedure, and Why CLSI No Longer Recommends It","MHT detects carbapenemase via a clover-leaf growth pattern, and CLSI dropped it from its guidelines in 2018 in favor of mCIM and Carba NP. The procedure, the limitations that led to its replacement, and where it's still used today.","2015-01-06",[],[46],{"slug":115,"title":116,"description":116,"seoTitle":37,"seoDescription":37,"author":58,"createdDate":117,"lastUpdatedDate":40,"draft":41,"category":42,"image":37,"faq":118,"tags":119},"stokes-disc-diffusion-method-principle-procedure-interpretation-results","Stokes Disc Diffusion Method: Principle, Procedure, Results","2014-12-28",[],[46],{"slug":121,"title":122,"description":123,"seoTitle":37,"seoDescription":37,"author":58,"createdDate":124,"lastUpdatedDate":125,"draft":41,"category":42,"image":37,"faq":126,"tags":145},"minimum-inhibitory-concentration-mic-broth-dilution-method-procedure-interpretation","Broth Dilution Method for MIC: Macrodilution vs Microdilution, Procedure & Troubleshooting","Step-by-step broth macrodilution and microdilution procedure for MIC determination — antibiotic stock prep, 0.5 McFarland standardization, reading results, and a troubleshooting guide for the errors that actually happen at the bench.","2013-11-15","2026-07-19",[127,130,133,136,139,142],{"question":128,"answer":129},"What's the difference between broth macrodilution and microdilution?","They follow the same principle — serial antibiotic dilutions inoculated with a standardized bacterial suspension — but macrodilution uses 1 mL per tube while microdilution uses 0.05–0.1 mL per well in a 96-well tray. Microdilution is far more common in routine clinical labs.",{"question":131,"answer":132},"Why must the inoculum be standardized to a 0.5 McFarland standard?","The MIC result depends directly on how many bacteria you start with. Too light an inoculum reads a falsely low MIC; too heavy reads a falsely high one. The 0.5 McFarland standard ensures every test starts from a comparable, known bacterial density.",{"question":134,"answer":135},"Why are reference strains like E. coli ATCC 25922 run alongside patient isolates?","They're quality-control checks. If a known reference strain's MIC falls outside its established CLSI range, it signals a problem with the test itself — inoculum, media, or technique — before patient results are trusted.",{"question":137,"answer":138},"What happens if microdilution trays are stacked too high during incubation?","Cultures at the center of a tall stack can incubate at a different temperature than intended, skewing growth and MIC readings — CLSI guidance caps stacking at four trays high.",{"question":140,"answer":141},"Can broth dilution MIC testing be automated?","Yes — systems like Vitek 2, MicroScan Walkaway, and BD Phoenix automate broth microdilution and reading, and are widely used in clinical labs alongside or instead of manual testing.",{"question":143,"answer":144},"Why might an MIC come back lower than clinically expected even with correct technique?","A few possibilities: the inoculum was too light, the cation-adjusted Mueller-Hinton broth's pH or calcium concentration is off, or there's a transcription\u002Freading error.",[46],{"slug":147,"title":148,"description":148,"seoTitle":37,"seoDescription":37,"author":58,"createdDate":149,"lastUpdatedDate":40,"draft":41,"category":42,"image":37,"faq":150,"tags":151},"basic-sets-drugs-used-routine-antimicrobial-susceptibility-testing-ast","Antimicrobial Susceptibility Testing (AST): Guidelines and Best Practices","2013-08-27",[],[46],{"slug":153,"title":154,"description":155,"seoTitle":37,"seoDescription":37,"author":58,"createdDate":156,"lastUpdatedDate":40,"draft":41,"category":42,"image":37,"faq":157,"tags":176},"interpretation-susceptibility-testing-susceptible-intermediate-resistant-mean","Susceptible, Intermediate, or Resistant: What the Categories Actually Mean (and Why \"Intermediate\" Isn't a Maybe)","S\u002FI\u002FR isn't a fixed mm or μg\u002FmL cutoff — it's organism-and-drug-specific. Learn how breakpoints are set, why \"Intermediate\" reflects real pharmacology rather than uncertainty, and a worked clinical example.","2013-08-26",[158,161,164,167,170,173],{"question":159,"answer":160},"Is a zone diameter of 20mm always \"Susceptible\"?","No. Breakpoints are specific to each organism-drug combination. The same 20mm zone can be Susceptible for one pairing and Resistant for another — always check the correct CLSI or EUCAST breakpoint table for that exact isolate and drug.",{"question":162,"answer":163},"Does \"Intermediate\" mean the antibiotic might not work?","Not automatically. It means the drug is only reliably effective in specific situations — at infection sites where it concentrates above serum levels (like urine), or at a higher approved dose. Outside those situations, response rates are genuinely lower than for Susceptible isolates.",{"question":165,"answer":166},"What's the difference between Intermediate and Susceptible-Dose Dependent (SDD)?","Intermediate reflects a buffer zone with conditional effectiveness depending on site or dosing flexibility. SDD specifically means effectiveness depends on using the higher end of the approved dosing range — it's a more defined, dose-driven category introduced more recently by CLSI.",{"question":168,"answer":169},"Why does CLSI use Nonsusceptible (NS) instead of Resistant for some results?","NS is used when no resistant strains of that organism have yet been documented for that drug, so only a Susceptible breakpoint exists. A result above that breakpoint is flagged Nonsusceptible rather than confidently called Resistant.",{"question":171,"answer":172},"Why is benzylpenicillin against staphylococci reported only as Susceptible or Resistant?","Because resistance in this pairing corresponds directly to β-lactamase production — an all-or-nothing mechanism — so there's no clinically meaningful intermediate zone.",{"question":174,"answer":175},"Do CLSI and EUCAST always agree on breakpoints?","Not always. CLSI (used widely in the US and South Asia) and EUCAST (the European standard) sometimes set different breakpoints for the same drug-organism pair, based on differences in dosing practices and population MIC data — always confirm which system a given lab report is using.",[46],[178,184,191,196,200,204,209,214,218,222],{"slug":179,"name":58,"description":180,"image":181,"body":182,"postCount":183},"acharya-tankeshwar","Editor-in-chief","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Ftankeshwar-acharya-author-microbeonline.jpg","***Tankeshwar Acharya, MSc (Medical Microbiology)***\n\n*Tankeshwar Acharya is an Assistant Professor in the Department of Microbiology at Patan Academy of Health Sciences (PAHS), Nepal, where he has been teaching and practicing clinical microbiology for over 14 years. He is the founder of Microbe Online, one of the leading free microbiology education resources on the web, covering bacteriology, mycology, parasitology, immunology, and clinical laboratory diagnostics written from direct experience in both the classroom and the diagnostic laboratory.*",433,{"slug":185,"name":186,"description":187,"image":188,"body":189,"postCount":190},"ashma-shrestha","Ashma Shrestha","SEO Copywriter and Science Communicator\nKathmandu, Nepal","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Fashma-shrestha.png","Ashma Shrestha holds a Master of Science in Medical Microbiology from the Institute of Science and Technology (IOST), Tribhuvan University, Nepal, where she developed a strong foundation in virology, molecular biology, and diagnostic microbiology.\n\nShe now works as an SEO Copywriter at Resolution Digital, where she combines her scientific training with research-driven content strategy. She is certified in Google Analytics and Google Business Profile (GBP), and brings a data-informed approach to science communication writing content that is not only accurate but structured to reach and serve the students who need it most.\n\nAt microbeonline, Ashma contributes articles primarily in virology and molecular biology, areas she finds most compelling for their mechanistic depth and their growing clinical relevance. Her writing reflects the same standard the site is built on: factual rigor, clear explanation of the *why* behind microbiology concepts, and content that helps students move from memorization to genuine understanding.\n\nShe is passionate about making complex microbiological concepts accessible without sacrificing accuracy; a skill that sits at the intersection of her scientific training and her professional work in content and SEO.",81,{"slug":192,"name":193,"description":194,"image":37,"body":37,"postCount":195},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":197,"name":198,"description":194,"image":37,"body":37,"postCount":199},"samikshya-acharya","Samikshya Acharya",20,{"slug":201,"name":202,"description":194,"image":37,"body":37,"postCount":203},"alisha-tripathi","Alisha Tripathi",6,{"slug":205,"name":206,"description":207,"image":37,"body":37,"postCount":208},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",9,{"slug":210,"name":211,"description":212,"image":37,"body":37,"postCount":213},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":215,"name":216,"description":194,"image":37,"body":37,"postCount":217},"srijana-khanal","Srijana Khanal",18,{"slug":219,"name":220,"description":212,"image":37,"body":37,"postCount":221},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":223,"name":38,"description":194,"image":37,"body":224,"postCount":225},"nisha-rijal","**Nisha Rijal** is a microbiologist and quality assurance specialist. She served for nearly 12 years as a microbiologist at the National Public Health Laboratory (NPHL), Nepal's national reference laboratory, and continues to work as a consultant microbiologist in international public health organization. ",51]