Antimicrobial resistance detection

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Laboratory methods to detect Antimicrobial Resistance:

Laboratory methods to detect Antimicrobial Resistance By Dr. Lavanya Giridhar


Aim The primary goal of antimicrobial susceptibility testing is to determine whether the bacterial etiology of concern is capable of expressing resistance to the antimicrobial agents that are potential choices for the therapy.

Historical milestones:

Historical milestones Beijerinck 1889- agar diffusion for auxins on bacterial growth Fleming in 1920- penicillin, ditch plate technique, broth macrodilution Heatley 1940- antibiotic discs Schmith & Reymann- agar dilution method Ericsson & Sherris- breakpoint techniques Bauer Kirby 1966. - Disk diffusion testing


Terms MIC Minimum inhibitory concentration is defined as the lowest concentration of antimicrobial agent that inhibits visible growth. MBC Minimum bactericidal concentration is defined as the lowest concentration of antimicrobial agent that kills 99.9% of the original inoculum.


Breakpoint Breakpoint, in its simplest terms, represents the concentration of an antimicrobial that separates populations of microorganisms. Vary with time, area Result--susceptible/ intermediate/resistant Depend on concentration of drug achieved in vivo at a standard dose


Interpretation Susceptible – inhibition at routinely achieved concentration Intermediate - inhibition only in sites where physiological concentration of antibiotic occurs or higher concentration of drug can be used Resistant - not inhibited


Terms Pharmacodynamics What drug does to the body (drug effects) Mechanisms of drug action, dose effect relationship Pharmacokinetics What body does to the drug (drug movement in ,through and out of the body) Absorption, distribution, metabolism, excretion, kinetics of elimination

Factors affecting distribution of drug in vivo :

Factors affecting distribution of drug in vivo Antibiotic diffusion in tissues Serum protein binding Drug interactions and interference Immune status of patient Virulence & pathogenicity of infecting bacterium Site & severity of infection

Differences between in vitro and in vivo drug action :

Differences between in vitro and in vivo drug action Factors In vitro In vivo Drug concentration fixed varying Protein binding absent present Time for drug exposure 16 to 24 hours Days for treatment courses Physiological conditions(eg pH) fixed variable Host immune response absent present Growth conditions optimum restricted Inocula at the start of drug exposure low high

Selection of antimicrobial agents for testing::

Selection of antimicrobial agents for testing: The antimicrobial agents that are chosen for testing against a particular bacterial isolate are referred to as the antimicrobial battery or panel in accordance with the CLSI guidelines Selection of most appropriate antimicrobial agents to test and to report is a decision best made by each clinical laboratory in consultation with the infectious disease practitioners and the pharmacy. The selection of an antibiotic panel for susceptibility testing is based on the commonly observed susceptibility patterns , and is revised periodically.

Classification of antibacterial agents according to Sites of action:

Classification of antibacterial agents according to Sites of action Cell wall synthesis Cell membrane function Protein synthesis Nucleic acid synthesis Metabolism Others

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Beta Lactam Antibiotics :

Beta Lactam Antibiotics Natural Penicillin PENICILLIN G Semisynthetic Penicillin Penicillinlinase producing Extended spectrum Aminopenicillins Carboxypenicillins Ureidopenicillins 3.  lactamase inhibitors

Cephalosporins :


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All Cephalosporins have 7-amino cephalosporinic acid (7-ACA) ring attached in their structure. Later generations came in existence by the difference in the nucleus of 7-ACA side chain. This modification also gave birth to Cephamycin group which includes Cefoxitin and Cefotetan , which are resistant to many β-lactamases.

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MONOBACTAMS Eg. Aztreonam the β-lactam ring is alone and not fused to another ring (in contrast to most other β-lactams, which have at least two rings). They work only against Gram-negative bacteria

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CARBAPENEMS Eg. Imipenem, Meropenem, Doripenem Recently, alarm has been raised over the spread of drug resistance to carbapenem antibiotics among these coliforms, due to production of the New Delhi metallo-β-lactamase, NDM-1 . There are currently no new antibiotics in the pipeline to combat bacteria resistant to carbapenems, and worldwide spread of the resistance gene is considered a potential nightmare scenario.


GLYCOPEPTIDES Vancomycin is added to the bacterial environment while it is trying to synthesize new cell wall. Here, the cell wall strands have been synthesized, but not yet cross-linked. Binds to the two D-ala residues on the end of the peptide chains. However, in resistant bacteria, the last D-ala residue has been replaced by a D-lactate, so vancomycin cannot bind. In resistant bacteria, cross-links are successfully formed, in the sensitive bacteria, the vancomycin bound to the peptide chains prevents them from interacting properly with the cell wall cross-linking enzyme. In the resistant bacteria, stable cross links are formed. In the sensitive bacteria, cross-links cannot be formed and the cell wall falls apart

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Tetracyclines :

Tetracyclines inhibiting the binding of aminoacyl t RNA to the mRNA-ribosome complex. They do so mainly by binding to the 30S ribosomal subunit in the mRNA translation complex.

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Aminoglycoside Amikacin Gentamicin Kanamycin Tobramycin Streptomycin

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Antimicrobial Susceptibility Testing :

Antimicrobial Susceptibility Testing

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Group A routine, primary testing panel Group B primary testing. reported selectively, when resistance to group A . Group C alternative or supplemental antimicrobial agents if strains resistant to primary drugs & also for selective reporting Group U (Urine) used only or primarily for treating urinary tract infections. A B C U Ampicillin Cefazolin Gentamycin Tobramycin Amikacin Amoxicillin clavulinic acid Ampicillin sulbactum Cefuroxime Ciprofloxacin imipenem Aztreonam Ceftazidime Chloramphenicol Tetracycline Cephalothin Lomefloxacin Ofloxacin Norfloxacin Nitrofurantoin Sulfisoxazole trimethoprim

Factors Influencing Antimicrobial Susceptibility Testing:

Factors Influencing Antimicrobial Susceptibility Testing pH Cation concentration Atmosphere Temperature Inoculum Pharmacokinetics

1.pH :

1.pH The pH of each batch of Mueller-Hinton agar should be checked when the medium is prepared by pH meter. The agar medium should have a pH between 7.2 and 7.4 at room temperature. If the pH is acidic, potency of certain drugs is affected e.g. Aminoglycosides and Macrolides Tetracycline

2. Cation concentration:

2. Cation concentration Ca++ & Mg++ aminoglycosides effect concentration of Ca++ is critical to the interpretation of daptomycin susceptibility zinc carbapenems Oxacillin resistance can be detected better when 2% NaCl present in media

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3.Atmosphere - ambient air incubator CO 2 incubator not used routinely. 4.Temperature - routinely at 35 o C, But certain conditions like OXACILLIN resistance  at 30 o C 5. Inoculum - some organisms contain inducible enzymes ( Eg - penicillins and betalactam antibiotics) - Hetero resistance- as small no. of bacteria express resistance to a particular drug so if the inoculum is less than there are chances that organism may not present in the inoculum . ( Eg – Methicillin and Staphylococci) Note : Homoresistance – Uniform expression of all organisms. Enterococci are inherent resistant to Aminoglycosides and Cephalosporins .

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6. Pharmacokinetics – Eg – Chloramphenicol is excreted via billiary tract Norfloxacin and Nitrofurantoin via urinary tract  Also ineffectiveness of Aminoglycosides to Legionella despite good sensitivity due to poor penetration in side macrophages 1 st and 2 nd generation Cephalosporins , Macrolides , Flouroquinolones do not penetrate in to CSF or donot accumlate in theraputic concerntration

McFarland turbidity standard:

McFarland turbidity standard The 0.5 McFarland standard, density of a bacterial suspension of 1.5 x 10 8 colony forming units (CFU)/ml. prepared by 0.5-mL aliquot of 1.175% w/v BaCl2 · 2H2O added to 99.5 mL of 1% w/v H2SO4 . The absorbance at 625nm should be 0.008 to 0.10 for 0.5 McFarland standard.

Antimicrobial Susceptibility on Solid Media:

Antimicrobial Susceptibility on Solid Media Relies on the use of agar as the solidifying agent and the nature of agar , which permits slow diffusion of antibiotics through its three-dimensional matrix. There are now three formats for testing using solid media: disk diffusion, agar dilution and gradient diffusion( E Test)

Special testing media:

Special testing media N. gonorrhoea- GC agar base Streptococcus spp.- MHA with 5% sheep’s blood N. meningitidis-MHA with 5% sheep’s blood H. influenza- Haemophilus Test Medium Burkholderia, B.anthracis, Yersinia- cation adjusted MHA

Methods of Antimicrobial Susceptibility Testing :

Methods of Antimicrobial Susceptibility Testing Disk Diffusion Dilution Dilution And Diffusion.


DISK DIFFUSION SUSCEPTIBILITY TESTING based on the diffusion through agar of drug released from an impregnated disk. zone diameter depends on disk content agar depth factors influencing the zone sizes disk content i.e amount of drug in the disk disk size diffusion characteristics of the drug depth of the agar growth rate of the bacterium (including the initial lag phase) activity of the drug against the strain being tested.

Preparation of dried filter paper discs:

Preparation of dried filter paper discs Wattman filter paper no. 1 6mm discs- sterilized in a hot air oven The loop used for delivering the antibiotics is made of 20 gauge wire and has a diameter of 2 mm. This delivers 0.005 ml of antibiotics to each disc. Stock solutions are prepared using the formula: 1000/P* V*C= W P-potency of anitbiotic base V= volume in ml reqd C=final conc. of solution W=weight of the antimicrobial to be dissolved in V. Refrigerate the containers at 8  C or below, or freeze at -14  C or below, in a nonfrost-free freezer until needed

Kirby Bauer disk diffusion susceptibility testing:

Kirby Bauer disk diffusion susceptibility testing Once isolated colonies are available from an organism that has been identified as a potential pathogen, it is necessary to proceed as follows to perform the susceptibility test. 1. Select colonies 2. Prepare inoculum suspension 3. Standardize inoculum suspension 4. Inoculate plate 5. Add antimicrobial disks 6. Incubate plate 7. Measure inhibition zones 8. Interpret results

Colony selection:

Colony selection bailey & scott's diagnostic microbiology 12th edi

Removing excess liquid from the swab:

Removing excess liquid from the swab bailey & scott's diagnostic microbiology 12th edi

Inoculating the Plate :

Inoculating the Plate bailey & scott's diagnostic microbiology 12th edi

Applying the Antimicrobial Disks :

Applying the Antimicrobial Disks Disc should be 15 mm from the edge of the plate and no closer than 25 mm from disc to disc

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bailey & scott's diagnostic microbiology 12th edi Disk diffusion test Before incubation After incubation

Measuring Zones:

Measuring Zones Reflected Light Reflected light is used for Enterobacteriaceae , such as E. coli , other gram-negative bacilli, s taphylococci and e nterococci (except for oxacillin and vancomycin). • Reflected light also is used when measuring zones on blood MHA (BMHA) Transmitted Light Use transmitted light, rather than reflected light, when measuring zones for: Staphylococci with o xacillin Enterococci with v ancomycin

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Reflected light is used to measure zones from back of plate:

Reflected light is used to measure zones from back of plate

Measuring zones on a blood agar plate with lid off:

Measuring zones on a blood agar plate with lid off


INTERPRETING RESULTS Measuring Unusual Zones: -Double zone: Measure the innermost zone. - Colonies within the zone: This can be due to either a mixed culture, which usually is obvious, or a resistant subpopulation of the test bacterium.

Interpreting Zone with Feathered Edge:

Interpreting Zone with Feathered Edge Measure the point at which you can see an obvious demarcation between growth and no growth. Avoid straining to see the tiniest colonies .

Swarming due to Proteus mirabilis :

Swarming due to Proteus mirabilis Measure the obvious zone. Ignore the swarm even if it covers the zone.


Trimethoprim-Sulfamethoxazole This agent may not inhibit bacteria from multiplying until the bacteria have gone through several generations of growth. light haze of growth may be seen within the zone. Measure the zone at the point where there is an 80% reduction in growth.

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Advantages: 1. Convenience & user friendliness 2. More antimicrobial agents can be tested against one bacterial isolate with minimal use of extra materials & devices 3. Relatively accurate results 4. Commonly encountered bacteria reliably tested Disadvantages: 1. Inability to provide more precise data regarding the level of an organism’s resistance or susceptibility as can be provided by MIC methods. 2. Lack of interpretative data for less common organisms.

Stokes method:

Stokes method Sensitive – Zone size equal to, wider than or not more than 3mm smaller than control Intermediate – Zone diameter greater than 3mm but smaller than control by more than 3mm Resistant – Zone diameter 3mm or less Control Organism Test Organism Antibiotic Disc

Modifications :

Modifications 3D testing -Antibiotic reservoirs consisting of antibiotic solutions in agar wells or cylinders Ericsson method- Inoculation by flooding method leading to semiconfluent growth Rolinson’s method - Plastic tray: 4 rows of 8 wells (each 10x15mm and 3 mm deep) filled with agar. Disc with antibiotic placed for diffusion of drug (min 3 hrs) Agar inoculated with swab dipped in suspension and see for growth after overnight incubation.

Agar Dilution susceptibility testing:

Agar Dilution susceptibility testing antimicrobial agent incorporated into the agar medium with each plate containing a different concentration of the agent. inocula applied using an inoculum replicating apparatus transferring 32–36 inocula to each plate ideal for regional reference laboratories or research laboratories that must test large numbers of isolates .

Agar dilution derivative:

Agar dilution derivative Instrument used to apply antimicrobial agent to the surface of an already prepared agar plate in a concentric spiral fashion Starting in the centre of the plate, the instrument deposits the highest concentration of antibiotic and from that point drug application proceeds to the periphery of the plate.


GRADIENT DIFFUSION SUSCEPTIBILITY TESTING The E test uses plastic strips; one side with the antimicrobial agent concentration gradient and the other with a numerical scale that indicates the drug concentration. Following overnight incubation, the number present at the point at which the border of growth inhibition intersects the E strip is taken as MIC. Several strips used to test multiple antimicrobials simultaneously

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Antibiotic gradient is created on the strip by applying different concentrations of antibiotics in repeated arrays of an increasing number of small dots.

Susceptibility Testing of Antimicrobials in Liquid Media:

Susceptibility Testing of Antimicrobials in Liquid Media Broth macrodilution method involves inoculation of a suspension of the organism into a liquid growth medium that incorporated serial twofold dilutions of the antibiotic to be tested. Initially, this technique was performed in Modified as the microdilution technique

Factors that Modify the Results of Broth Dilution Tests :

Factors that Modify the Results of Broth Dilution Tests Dilution schedule Serial twofold vs arithmetic intervals Medium composition pH Cation concentration Osmolarity Supplements Volume Inoculum size and growth phase Temperature of incubation Duration of incubation Varying quality control and standardization procedures

Broth macrodilution:

Broth macrodilution Serial dilutions of antimicrobial agent made in MH broth to which a standardized bacterial suspension is added. Final volume of 1 to 2 mL per test tube. After incubation at 35 0 C for 18 hrs, tubes are visually examined for turbidity. 1 st clear tube is the MIC.

Broth micro dilution methods:

Broth micro dilution methods Broth micro dilution MIC testing is performed in a polystyrene panel containing approximately 96 wells. Panel may contain 7–8 dilutions of 12 different antimicrobial agents and includes positive & negative growth control. volume –up to 0.5 mL in each well. Mueller-Hinton broth is recommended

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Growth indicated by turbidity or a button of growth >2mm Negative wells should be clear MIC endpoint is read as the lowest concentration of antimicrobial agent that completely inhibits growth of the organism as detected by the unaided eye TRAILING

Breakpoint MIC testing:

Breakpoint MIC testing One or two concentrations of a antimicrobial agent tested Results- susceptible/ resistant Adv- Endpoints clear, no trailing Disadv- Precise MIC not obtained


Advantages Small volumes of reagents required Large number of isolates can be tested Automated dispensing of known dilution of antibiotics Frozen plates or lyophilised plates available commercially Automated readers available

Comparative Feature of Disk Diffusion and Dilution Methods :

Comparative Feature of Disk Diffusion and Dilution Methods Feature DDST Agar dilution Broth dilution Contamination readily observed Y Y N Inoculum effect N N Y Quantitative result N Y Y Direct test from clinical material Y N Applicable for slow-growing and fastidious organisms N Y Y Readily amenable to automation N Y

Automated antimicrobial susceptibility test systems:

Automated antimicrobial susceptibility test systems The automated antimicrobial susceptibility test systems include the bioMerieux Vitek 2 systems, Dade Microscan walk away system and the BD phoenix system Conventional testing is performed using colorimetric and turbidity readings. The rapid-testing system utilizes fluorogenic substrates and fluorometric indicators.

Nucleic acid amplification test (PCR):

Nucleic acid amplification test (PCR) staphylococcus mecA ant erm,msr gyrA Methicillin Aminoglycocides Macrolides fluoroquinolones Probe,PCR Probe Probe,PCR Probe,PCR S. pneumoniae pbp1A,2B,2X b-lactams PCR Enterococcus spp Van A,B,C aac,aph, ant Glycopeptides Aminoglycocides Probe,PCR Probe,PCR

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Enteobacteriaceac tem,shv aph , ant, aac cat tet erm,ere Dhfr vim ,imp b-lactams Aminoglycocides Chloramphenicol Tetracycline Macrolides Trimethoprim carbapenems Probe,PCR Probe,PCR Probe Probe,PCR Probe,PCR Probe PCR H.influenzae tem b-lactams Probe,PCR

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OXA-type -lactamases - oxacillin-hydrolyzing abilities CTX -hydrolytic activity against cefotaxime TEM-1 was first reported in 1965 from an Escherichia coli isolate from a patient in Athens, Greece, named Temoneira SHV refers to s ulf h ydryl v ariable. VEB – 1 st isolate 4m Vietnamese infant PER-1 -first detected in Pseudomonas aeruginosa Toho-1 -Toho University School of Medicine Omori Hospital in Tokyo CME-1- from Chryseobacterium meningosepticum SFO-1- Serratia fonticola VIM-1 (for “Verona integron-encoded metallo--lactamase”) IMP-1 (for “active on imipenem”), SPM-1 (for “Sao Paulo metallo--lactamase”) GIM-1 (for “German imipenemase”) SIM-1 (for “Seoul imipenemase”) bailey & scott's diagnostic microbiology 12th edi

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DRUG RESISTANCE Intrinsic or natural Resistance e.g. vancomycin and penicillin vs. GNB Acquired Resistance Development of resistance over a period. Possible mechanisms are : Mutation single step multi step 2. Gene transfer conjugation Transduction Transformation

Intrinsic resistance:

Intrinsic resistance Salmonella spp., Shigella spp. 1 st & 2 nd gen cephalosporins, cephamycins, aminoglycosides Enterococcus spp., Aminoglycosides , cephalosporins , clindamycin , trimethoprim-sulfamethoxazole Yersinia pestis b- lactam

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Emergence of resistance Mixing of bacterial gene pool Selective pressure from excessive antimicrobial use and abuse Survival of the fittest

Detection of specific resistance mechanisms:

Detection of specific resistance mechanisms B lactamases ESBL AmpC Carbapenamases Oxacillin resistance staphylococci Vancomycin resistance HLAR

β lactamases:

β lactamases Visual detection of end products of β lactamase hydrolysis which is demonstrated by colorimetric reaction Method Substrate Reaction Nitrocephin Nitrocefin Color change to red when b lactam ring opens Acidometric Citrate buffered penicillin + phenol red Penicilloic acid produces pH Iodometric phosphate buffered penicillin + starch iodine complex Penicilloic acid reduces iodine prevents it from combining with starch

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MRSA Oxacillin and Methicillin resistant Resistant to all beta-lactam agents, including cephalosporins and carbapenems. MRSA isolates are often multiple resistant to commonly used antimicrobial agents, including erythromycin, clindamycin, and tetracycline.


MRSA PBPs are membrane bound enzymes that catalyses transpeptidation reaction that is necessary for cross linkage of pepidoglycan chains. MRS - +nce of mec A gene  responsible for synthesis of PBP2a , substitute for PBP which has low affinity for beta lactam antibiotics. Agar dilution- MHA with 4% NaCl & 6ug/ml oxacilliin, 33 0 C, 24 hrs, transmitted light Glycopeptides, Vancomycin and Teicoplanin are the only drug of choice for treatment of severe MRSA infections, although some strains remain susceptible to fluoroquinolones, trimethoprim/sulfamethoxazole, gentamicin

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Oxacillin maintains its activity during storage better than methicillin and is more likely to detect hetero - resistant strains. However, cefoxitin is an even better inducer of the mecA gene and disk diffusion tests using cefoxitin give clearer endpoints and are easier to read Cefoxitin is used as a surrogate Cefoxitin ≤ 21 mm - mecA positive Cefoxitin ≥ 22 mm - mecA negative


CONT. Isolates that tests as mecA positive should be reported as oxacillin (not cefoxitin ) resistant. Other beta lactam agents should be reported as resistant or should not be reported rare occurrence of resistance mechanisms other than mecA , isolates that test as mecA negative, but for which the oxacillin MICs are resistant (MIC ≥ 4μg/m) ,should be reported as oxacillin resistant.


ESBL DETECTION R extended to 3 rd generation cephalosporins . (Cefotaxime, Ceftazidime , Ceftraixone ) and Monobactams BUT do not affect cephamycins ( Cefoxitin , Ceotetan ) and Cabepenems ( Imipenem , Meropenem )  choice of which antimicrobial agents to test is critical. Eg . one enzyme may actively hydrolyze ceftazidime , resulting in ceftazidime minimum inhibitory concentrations (MICs) of 256 μg /ml, but have poor activity on cefotaxime , producing MICs of only 4 μg /ml.  If an ESBL is detected, all penicillins , cephalosporins , and aztreonam should be reported as resistant, even if in vitro test results indicate susceptibility.

Note :

Note When using new interpretative criteria (CLSI, 2011) routine ESBL testing is no longer necessary before reporting results (i.e. it is no longer necessary to edit the results of cephalosporins , aztreonam or penicillin to resistant). However, until laboratories implement the new interpretative criteria, ESBL testing should be performed. ESBL testing may still be useful for infection control and epidemiological surveillance.

Screening test :

Screening test For Klebsiella pneumoniae, Klebsiella oxytoca & Escherichia coli Cefpodoxime (10 μ g) Ceftazidime (30 μ g) Aztreonam (30 μ g) Cefotaxime (30 μ g) Ceftriaxone (30 μ g) For Proteus mirabilis Cefpodoxime (10 μ g) Ceftazidime (30 μ g) Cefotaxime (30 μ g)

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The use of more than one of the five antimicrobial agents suggested for screening will improve the sensitivity of detection. Cefpodoxime and ceftazidime show the highest sensitivity for ESBL detection

Confirmatory tests:

Confirmatory tests Disk potentiation Double disk synergy test 3D test E test

Disk potentiation:

Disk potentiation Disk diffusion Ceftazidime/ cefotaxime 30ug Ceftazidime/ cefotaxime + clavulanic acid 30ug+ 10ug Difference of >= 5 mm Broth microdilution Ceftazidime/ cefotaxime Ceftazidime/ cefotaxime + clavulanic acid >3 twofold decrease in MIC Dumbbell shaped

Double disk synergy test :

Double disk synergy test disk containing amoxicillin-clavulanate is placed disks containing cefotaxime/ ceftriaxone are placed 30 mm (center tocenter) from the amoxicillin-clavulanate disk. enhancement of the zone of inhibition caused by the synergy of the clavulanate in the amoxicillin-clavulanate disk is a positive result

3D test:

3D test Inoculation of the std organism on MHA plate A slit is cut into the agar, in which a broth suspension of the test organism introduced. antibiotic disks are placed on the surface of the plate 3 mm from the slit. Distortion/discontinuity in the expected circular zone of inhibition is considered a positive test

Disc Approximation Test:

Disc Approximation Test Cefoxitin (inducer) disc is placed at a distance of 2.5 cm from Cephalosporin disc. Production of inducible b-lactamase is indicated by flattening of the zone of inhibition of the Cephalosporin disc towards inducer disc by 1mm or more than that.

Metallo – β-lactamase (MBL):

Metallo – β- lactamase (MBL) Metallo –B-lactamase requires zinc for their catalytic activity. inhibited by Metal chelators, such as EDTA and THIOL compounds. hydrolyze all beta-lactam antibiotics including, penicillins, cephalosporins, and carbapenems, with the exception of aztreonam (monobactam). MBL producing strains are not susceptible to serine beta lactamase inhibitors,(e.g. clavulanate) .

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Plasmid mediated MBL genes spread rapidly to other species of gram negative bacilli; therefore rapid detection of metallo-β-lactamases production is necessary to modify therapy and to initiate effective infection control to prevent their dissemination. Since there is no standard guideline for detection of MBL, different studies have reported the use of different methods. Screening for MBL production is to be carried out in imipenem resistant isolates.

1. Imipenem- EDTA combined disc test (CDT). :

1. Imipenem - EDTA combined disc test (CDT). SCREENING TEST A 0.5 M EDTA solution was prepared adjusting its ph 8.0. Two imipenem (10ug) discs were placed on the surface of an agar plate at distance of 25 mm and 4 ul EDTA solution was added to one of them to obtain a desired concentration of 750 if the increase in inhibition zone with the imipenem and imipenem- EDTA disc was ≥7 mm than the imipenem alone, it was considered MBL positive

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Combined disc test

2.double- disc synergy test:

2.double- disc synergy test An imipenem (10ug) disc was placed 20 mm center to center from a blank disc containing 4 ul of ,0.5 M EDTA (750 ug). Positive result when enhancement of zone of inhibition between imipenem and EDTA disc ≥5mm

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3.Modified Hodge Test:

3.Modified Hodge Test detects carbapenemase production. Carbapenemase production is detected by the MHT when the test isolate produces the enzyme and allows growth of a carbapenem susceptible strain (E.coli ATCC 25922) towards a carbapenem disk. The result is a characteristic cloverleaf-like indentation.

AmpC detection:

AmpC detection AmpC β-lactamases, in contrast to ESBLs, hydrolyse broad and extended-spectrum cephalosporins ( cephamycins as well as to oxyimino-β-lactams) & are not inhibited by β-lactamase inhibitors such as clavulanic acid AmpC disc test 3D enzyme extraction test Cefoxitin agar medium Boronic acid disc test Modified Hodge test

AmpC disc test:

AmpC disc test Lawn culture of std E.coli on MHA AmpC disc (i.e Tris EDTA ) with test strain inoculated on them placed 30ug cefoxitin disc placed almost touching Flattening of zone positive test

3D enzyme extraction test:

3D enzyme extraction test Lawn culture with std E.coli 30ug cefoxitin disc placed Slit made 5 mm from disc with test strain enzyme inoculated Enhanced zone of growth-positive

Cefoxitin (screen) agar medium :

Cefoxitin (screen) agar medium Lawn culture of E.coli on MHA plate with cefoxitin 4ug/ml Test strain inoculated in well cut in agar Positive if growth around well

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Boronic acid disc test :

Boronic acid disc test Boronic acids have long been known as AmpC inhibitors a blank disk placed near a -lactam disk or added to the-lactam disk for comparison with an unmodified –lactam disk 30ug cefotetan 30ug cefotetan + 400ug boronic acid Positive if zone difference greater than 5mm

Carbapenem resistance:

Carbapenem resistance Acquires a carbapenemase enzyme or when an isolate produces an extended spectrum cephalosporinase, such as an Ampc – type Beta-Lactamase , in combination with porin loss. The most common mechanism of carbapenem resistance is the Klebsiella pneumoniae Carbapenemase (KPC).

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R to one or more carbapenems (Ertapenem non susceptibility is the most sensitive indicator of carbapenemase production) and one or more agents in cephalosporin subclass III (e.g. cefoperazone, cefotaxime, ceftazidime, ceftizoxime and ceftriaxone) .

Screening test for carbapenemase detection :

Screening test for carbapenemase detection Disk diffusion Ertapenem (10 μ g) OR Meropenem (10 μ g) Note : Imipenem disk performs poorly as a screen for carbapenemases

Carbapenamase detection:

Carbapenamase detection Modified Hodge test E test

Modified Hodge test:

Modified Hodge test Lawn culture of E.coli ATCC 25922 Isolate streaked towards meropenem disc 10ug in centre Enhanced growth positive for carbapenamase production Clover leaf shape

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Some test isolates may produce substances that will inhibit growth of E.coli ATCC 25922 . When this occurs, a clear area will be seen around the streak and the modified Hodge test is uninterpretable for these isolates For isolates positive with the ertapenem or meropenem disk screen & positive with the Modified Hodge test , perform MIC test before reporting any carbapenem results.

Inducible clindamycin drug resistance in staphylococcus spp.:

Inducible clindamycin drug resistance in staphylococcus spp. MLS B gene detection (constitutive or inducible) M - Macrolides L - Lincosamides S – Streptogramin B Methylation at 23s ribosomal subunit (target for M & L) [ enm A & enm C] Or efflux [ msr A] R may be only M type or LS type.

D test:

D test Resistance of weak inducer like clindamycin can be detected in presence of a strong inducer like erythromycin Erythromycin 30 ug Clindamycin 2 ug Disks 15-26 mm apart When both disc are plated separetly result would be R to E but S to C

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E C No resistance Inducible erm Constitutive erm Efflux E E E C C C

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Reporting of icr:

Reporting of icr Hazy growth within the zone of inhibition around clindamycin indicates clindamycin resistance, even if no D- zone is apparent. Report isolates with inducible clindamycin resistance as “ clindamycin resistant ”. A comment that “ This isolate is presumed to be resistant based on detection of inducible clindamycin resistance. Clindamycin may still be effective in some patients ” may be included.

Vancomycin resistance VRSA/VRE :

Vancomycin resistance VRSA/VRE Screening test by Disk diffusion Vancomycin (30 μ g) Interpretation Transmitted light. The presence of a haze or any growth within the zone of inhibition indicates resistance. A zone diameter of ≥ 17 mm is sensitive; 15-16 mm is intermediate, ≤ 14 mm is considered as resistant. Organism with intermediate zone should be tested by an MIC method.

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Confirmation by - MIC testing of Vancomycin By E test MIC ( μ g/ml) Interpretation ≤ 4 μ g/ml Sensitive 8-16 μ g/ml Intermediate ≥ 32 μ g/ml Resistant

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HLAR Gentamycin Disk diffusion-120ug Broth/ agar microdilution- 500ug/ml Agar dilution- 500ug/ml Streptomycin Disk diffusion-300ug Broth microdilution- 1000ug/ml Agar dilution- 2000ug/ml

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Resistant : is not synergistic with cell wall active agent (eg. Ampicillin, penicillin and vancomycin) Susceptible : is synergistic with cell wall active agent (eg. Ampicillin, penicillin and vancomycin) that is also susceptible. Inconclusive : perform an agar or broth microdilution test to confirm.


summary Screening Tests -characterize an isolate as susceptible or resistant to one or more antimicrobial agents based on a specific resistance mechanism or phenotype. -Some screening tests have sufficient sensitivity and specificity such that results of the screen can be reported without additional testing. Others require further testing to confirm the presumptive results obtained with the screen test. A summary of the some important screening tests is provided hereunder.


1. Staphylococcus aureus & Coagulase -negative Staphylococci  mecA -Mediated Oxacillin resistance Broth dilution and disk diffusion with Cefoxitin Further confirmation not required  Inducible Clindamycin resistance Broth microdilution and disk diffusion with clindamycin and erythromycin Further confirmation not required A summary of the some important screening tests

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2. Enterococci  Vancomycin resistance Agar dilution with vancomycin Yes, if screen test is positive  High Level Aminoglycoside resistance Broth microdilution, agar dilution and disk diffusion with gentamycin and streptomycin Further confirmation not required for MIC. Yes for disk, if inconclusive

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3. Enterobacteriaceae  ESBL Production Broth microdilution and disk diffusion with various cephalosporins and aztreonam Further confirmation required  Carbapenemase production Broth microdilution and disk diffusion with various cephalosporins Further confirmation required

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4. Pseudomonas aeruginosa & Acinetobacter species  Metallo- β- lactamases Imipenem-EDTA combined disk diffusion test


References CLSI manual of antimicrobial susceptibility testing Koneman – Diagnostic microbiology,6 th edition Manual on Antimicrobial Susceptibility Testing; Dr. M.K. Lalitha Mackie & McCartney practical medical microbiology, 14 th edition Bailey & Scott’s DIAGNOSTIC MICROBIOLOGY 12 th edition

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bailey & scott's diagnostic microbiology 12th edi Thank you thank you

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