|Extended-Spectrum Beta-Lactamase (ESBL) Activity|
Illustrated in the image is the surface of a disk diffusion test including a 30 mg ceftazidime disk (left) and a combintation 30/10 mg ceftazidime/clavulanic acid disk (right). Observe in the photograph that the zone of inhibition around the the combination ceftazidime/clavulanic acid disk (right) is at least 5 mm larger than around the clavulanic acid disk (left). This observation that the presence of clavulanic acid, a beta-lactamase inhibitor, has resulted in such a large increase in the zone of inhibition indicates that an extended-spectrum beta-lactamase (ESBL)is being produced. Additionally, the Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Testing Standards, published January 2011, proposes in the M100-S21document, table 2A-S1, that cefotaxime (30 mg) and cefotazime-clavulanic acid (30/10 mg) testing per performed alone AND in combination with the ceftazidime and ceftazidime/clavulanic acid testing previously described. When an organism is producing an ESBL, the susceptibility to individual cephalosporins cannot be predicted, thus requiring that each drug must be tested individually.
Vancomycin and ampicillin resistance among Enterococcus species, particularly E. faecium have been on a steady increase. The disk diffusion screening test is used in many laboratories to detect vancomycin resistant strains. Note in the upper image that no zone of inhibition is seen around either the vancomycin or the ampicillin disk, indicating resistance to both drugs. Vancomycin-resistant Enterococci (VRE) have been divided into three phenotypes--Van A, Van B, and Van C. Vancomycin-resistant strains of E. faecalis and E. faecium are commonly of the Van A phenotype, demonstrating high level resistance (MIC's higher than 64 ug/mL), as illustrated by total resistance of the test strain in the E test and the VA disk, as illustrated in the lower image. The strain shown in the lower image, however, is ampicillin susceptible at the level of 1 ug/mL (see lower set of yellow arrows), indicating that this drug may be effective in treating the urinary tract infection.
|Methicillin-Resistant Staphylococcus aureus (MRSA) Disk Test|
The disk diffusion test can also be used in the detection of methicillin-resistant Staphylococcus aureus. Illustrated in the image is the surface of a Mueller-Hinton agar plate previously inoculated with a strain of S. aureus suspected of being methicillin-resistant. Although the zone of inhibition is at the borderline for resistance (18 mm); the presence of small colonies within the zone of inhibition (yellow arrows) indicates the presence of heteroresistant strains. The interpretation here, therefore, is "methicillin-resistant" staphylococci, even though the zone diameter appears to be adequate. The detection of the heteroresistant strains indicates that minimum inhibitory concentration (MIC) studies are required.
|Susceptibility testing of Enterococci|
All susceptibility testing for Enterococci should follow the standards defined by CLSI. Selection of drugs for testing will follow the same criteria defined previously. Because the Enterococci posses many intrinisic resistance factors, there are many antibiotics that should not be tested (or if tested, suppressed from the final report). CLSI document M100 defines all applicable criteria. Both Disk Diffusion (Kirby Bauer) and broth dilution (MIC) methods are commonly employed, utilizing the following testing conditions: Medium: MHA for disk diffusion; CAMHB for broth dilution(supplemented to 50 ug/ml for daptomycin) Inoculum: Growth method or direct colony suspension, equivalent to a 0.5 McFarland standard Incubation: 35 + 2o C; ambient air. Disk diffusion; 16 to 18 hours; Dilution methods; 16 to 20 hours. All methods: 24 hours for vancomycin.
|Detecting Vancomycin Resistance|
All results for vancomycin should be interpreted according to CLSI criteria.Interpretive standards for Enterococcus species against Vancomycin Method Susceptible Intermediate Resistant Disk diffusion >17 mm 15 - 16 mm < 14 mm Broth dilution < 4 µg/mL 8 - 16 µg/mL > 32 µg/mL Screening methodologiesBHI agar with 6 µg/ml vancomycin can be employed as a screen for vancomycin resistance. 1 - 10 µl of a 0.5 McFarland standard suspension is spotted onto the agar surface and incubated at 35 + 2 o C for a full 24 hours. The presence of >1 colony equates to presumptive vancomycin resistance; these isolates would require species identification and a vancomycin MIC.A motility test and documentation of the presence or absence of a yellow pigment can distinguish between the species with intrinisic (VanC) resistance (gallinarum and casseliflavus) and those with acquired (VanA and VanB) resistance (faecium and faecalis).
High level resistance to aminoglycosides is another significant acquired resistance factor. Since the standard approach for treating systemic infections is a combination of a cell wall targeted antibiotic with an aminoglycoside, assessment of resistance to both classes of antibiotics is important. High level resistance to aminoglycosides will negate the synergistic effect of combined therapy with either penicillin or vancomycin.Standard susceptibility methods (either disk diffusion or broth dilution) will not detect HLR patterns, unless the protocol incorporates testing at increased concentrations of gentamicin and/or streptomycin. The CLSI documents outline recommended protocols for screening for HLR aminoglycoside resitance.Gentamicin HLARDisk diffusion: MHA agar; 120 ug gentamicin disc; standard inoculum and incubation temperature; incubation duration: 16 - 18 hours.Interpretation: Resistant = 6 mm Inconclusive = 7-9 mm Susceptible > 10 mmBroth microdilution: BHI broth; 500 ug/ml gentamicin; standard inoculum and incubation temperature, incubation duration: 24 hours.Interpretation: any growth equates to a resistant interpretation.Streptomycin HLARDisk diffusion: MHA agar; 300 ug streptomycin disc; standard inoculum and incubation temperature; incubation duration: 16 - 18 hours.Interpretation: Resistant = 6 mm Inconclusive = 7-9 mm Susceptible > 10 mmBroth microdilution: BHI broth; 1000 ug/ml streptomycin; standard inoculum and incubation temperature, incubation duration: 24 - 48 hours. If susceptible at 24 hours, reincubate and re-read at 48 hours.Interpretation: any growth equates to a resistant interpretation.Clinical correlationA resistant result indicates that synergistic effects will not be achieved between the indicated aminoglyocside and the cell wall active agent (eg, ampicillin, penicillin, or vancomycin).A susceptible result indicates that synergistic effects are possible.
|With regards to identifying resistance in Enterococci, which general statements are true?||View Page|
|Newer antibiotics for Treatment of Resistant Enterococci|
Synercid (quinupristin and dalfopristin) is an alternative therapy for systemic infections with VRE. It is FDA approved for treatment of life threatening infections with vancomycin resistant isolates of E. faecium. This antibiotic is appropriately suppressed from reports of other vancomycin sensitive strains of E. faecium, and all other species of Enterococcus.Linezolid belongs to the oxazolidinone class of antibiotics. It is active against VRE. The main indication of linezolid is treatment of severe infections caused by gram positive bacteria that are resistant to other antibiotics.Daptomycin is the first lipopeptide released onto the market. Daptomycin has been approved for the treatment of skin and soft tissue infections; evaluaton of efficacy in more serious systemic infections is ongoing. Disk diffusion testing for Daptomycin is not reliable.Telavancin is a lipoglycopeptide under trial for MRSA and other gram positive infections. The spectrum of activity of telavancin is similar to vancomycin, but it may be active against some VRE strains (Van B).
|Testing for Vancomycin Susceptibility|
The current CLSI recommendation is that MIC tests should be performed to determine the susceptibility of staphylococci to vancomycin. The disk test does not differentiate vancomycin-susceptible isolates of S. aureus from vancomycin-intermediate strains.Disk diffusion will detect S. aureus isolates containing the VanA vancomycin-resistance gene (VRSA). These isolates will show no zone of inhibition around the disk (zone = 6mm); their identification should be confirmed. Isolates producing vancomycin zones > 7mm should not be reported as susceptible without performing a vancomycin MIC test.Recommended methods are CLSI Broth Microdilution, Agar Dilution, and Etest® with inoculum prepared to match McFarland 0.5 turbidity standard. The Etest® is considered the most discriminatory of these methods as it allows for visualization of small colonies around zones of inhibition. A pure culture MUST be used. Repeat test for confirmation.The CLSI recommends that the inoculum should be prepared using the direct suspension method and plates incubated for a full 24 hours in ambient air at 35° C. Screening for vancomycin resistance in Staphylococci (MIC's > 8 ug/ml) can be performed utilizing a vancomycin agar screening plate – BHI (brain heart infusion) agar containing 6 mg/mL vancomycin. However testing on BHI screening agar does not reliably detect all vancomycin intermediate S. aureus strains.
|A laboratory's primary susceptibility testing method is disk diffusion. The cefoxitin disc has a zone size of 19 mm and the vancomycin disc has a zone size of 7 mm. Which of the following are appropriate courses of action?||View Page|
When a clinical isolate is presumptively identified as S. aureus, susceptibility testing will be performed by either the standardized disk diffusion (Kirby-Bauer) or broth dilution (MIC) methods, using the following testing conditions as recommended by the Clinical and Laboratory Standards Institute (CLSI):MediumMHA for disk diffusionCation-adjusted Mueller-Hinton broth (CAMHB) + 2% NaCL for oxacillin, methicillin, and nafcillinCAMHB supplemented up to 50 µg/mL calcium for daptomycin InoculumDirect colony suspension-- Inoculum from an 18-24 hour non-selective agar plate used to prepare a direct inoculum equivalent to a 0.5 McFarland Standard. Incubation35° C (Testing at temperatures above 35° C may not detect MRSA); 24 hours for oxacillin, methicillin, nafcillin, and vancomycin.
|Interpretation of Oxacillin and Cefoxitin Disk Diffusion Tests|
Oxacillin is the agent of choice for standardized MIC methods (broth & agar dilution). However, since 2006 the Clinical Laboratory Standards Institute (CLSI) has recommended the use of 30 µg cefoxitin disk rather than the oxacillin disk to detect mecA-mediated resistance in the disk diffusion test because the cefoxitin disk test is easier to read and is as sensitive and specific as MIC methods. Results are still reported as "oxacillin-resistant" or "oxacillin-sensitive." Cefoxitin is a better inducer of the mecA gene and gives clearer, easier to read endpoints in disk diffusion tests.The oxacillin disk is read for light growth within the zone of inhibition using transmitted light (plate held up to light), ANY discernible growth within zone of inhibition is indicative of resistance. The cefoxitin disk is read using reflected light.Interpretive Critieria for Cefoxitin Disk Diffusion TestResisitantIntermediateSusceptibleS.aureus/MRSA<21 mmN/A> 22 mm
|Detection of Oxacillin Resistance|
Resistance to oxacillin is most accurately determined by testing for mecA or for the protein expressed by mecA, the penicillin-binding protein 2a (PBP 2a, which is also referred to as PBP 2'). Isolates of staphylococci that carry the mecA gene, or that produce PBP 2a should be reported as oxacillin-resistant according to CLSI guidelines. If MIC tests are performed in addition to disk diffusion, isolates for which oxacillin MICs are > 4µg/mL, are mecA-negative, or PBP 2a negative should be reported as oxacillin-resistant. Such isolates may have a rare resistance mechanism other than mecA, and may also test susceptible to cefoxitin by disc diffusion. In these scenarios, oxacillin resistance should be reported in accordance with the MIC value.