Phenotypic detection of extended-spectrum beta-lactamases (ESBLs) is based on the inhibition of ESBL enzymes by β-lactamase inhibitors and on the comparison of cephalosporin activity with or without a β-lactamase inhibitor. Many South African diagnostic laboratories rely on the Vitek 2 for automated susceptibility testing and for ESBL detection. However, the Gram-negative susceptibility card currently used locally (AST-N255) has been modified and its accuracy for ESBL detection is not known.
We randomly selected 50 isolates of
The sensitivity of Vitek 2 system for detection of ESBLs was 33/36 or 92% (78% – 97%) for
Using a composite reference standard of the phenotypic and genotypic methods employed in this study, no Vitek-categorised ESBL
Extended-spectrum β-lactamases (ESBLs) are enzymes that hydrolyse most penicillins and cephalosporins, including oxyimino-β-lactam compounds, but not cephamycins or carbapenems.
As many ESBL-producing organisms are also resistant to other unrelated antibiotics, such as fluoroquinolones, aminoglycosides and trimethoprim-sulfamethoxazole, high rates of ESBL infections result in increased use of carbapenems, which, in turn, selects for carbapenem-resistant organisms, for which there are few therapeutic options.
In routine diagnostic laboratories, phenotypic methods for the detection of ESBLs are based on the inhibition of ESBL enzymes by β-lactamase inhibitors and on the comparison of cephalosporin activity with or without a β-lactamase inhibitor.
However, various modifications to the Gram-negative susceptibility card to meet local needs have necessitated the removal of the cephalosporin–clavulanic acid combination wells. Consequently, ESBL detection in South African laboratories is now based solely on the pattern of susceptibility and resistance to different cephalosporins. Experience with similarly modified Vitek 2 Gram-negative susceptibility cards elsewhere suggests that the specificity of ESBL detection may be reduced.
The aim of this study was therefore to determine the sensitivity and specificity of the Vitek 2 AST-N255 Gram-negative susceptibility card for the detection of ESBLs in
The Groote Schuur NHLS microbiology laboratory serves the southern part of the greater Cape Town area with a catchment population of approximately 2 million people. The laboratory receives approximately 3600 blood culture samples a month and from these maintains a stored collection of selected organisms. From the 2015–2016 collection of bloodstream isolates, we randomly selected 50 isolates of
Extended-spectrum β-lactamase production was detected by disc diffusion and broth microdilution methods, performed and interpreted according to CLSI criteria.
Disc diffusion testing was performed using the Kirby–Bauer method. Cefotaxime and ceftazidime discs with or without clavulanic acid were utilised, with an increase of ≥ 5 mm in zone inhibition diameter for either cephalosporin in the presence of the inhibitor, indicating the presence of an ESBL.
Broth microdilution was performed using the Sensititre™ ESBL plate format (Trek Diagnostic systems, ThermoScientific Waltham, MA, USA) in accordance with the manufacturer’s instructions. The Sensititre™ ESBL plate includes wells containing cefotaxime and ceftazidime with or without clavulanic acid. A ≥ 3 twofold concentration decrease in MIC for either cephalosporin in the presence of the inhibitor indicates the presence of an ESBL.
The ranges of MICs (in µg/mL) that can be determined using the Sensititre ESBL plate are ≤ 0.25–> 64, ≤ 0.12/4-–>64/4, ≤ 0.25–> 128 and ≤ 0.12/4–> 128/4 for cefotaxime, cefotaxime + clavulanic acid, ceftazidime and ceftazidime + clavulanic acid, respectively. An indeterminate result was reported when it was not possible to calculate the ratio accurately at the limits of MIC range, for example if cefotaxime MIC ≤ 0.25, and cefotaxime + clavulanic acid MIC ≤ 0.12/4. If an indeterminate result was obtained for either cefotaxime or ceftazidime, the isolate was categorised according to the result of the other antibiotic. If indeterminate results were obtained for both antibiotics, the ESBL status was determined by the MICs of the antibiotics, that is, if both cefotaxime and ceftazidime MICs were at the lower limit of the MIC range, the isolate was reported as ESBL negative, whereas if both MICs were at the upper limit of the range, the isolate was reported as ESBL-positive.
Bacterial DNA was extracted from colonies grown on 2% blood agar using the QiaSymphony SP automated extraction platform with the QiaSymphony DSP Virus/Bacteria mini kit, according to the manufacturer’s instructions (Qiagen, Hilden, Germany). To test for the presence of the most commonly occurring ESBL genes
The sensitivity and specificity of the Vitek 2 AST-N255 Gram-negative susceptibility card for ESBL detection was compared to a composite reference standard, in which an isolate was defined as an ESBL if either an ESBL was detected by both phenotypic methods, that is, by disc diffusion and by broth microdilution, or if an ESBL was detected by either phenotypic method as well as genotypically. The 95% confidence intervals for proportions were calculated according to Newcombe method.
Ethical clearance was obtained from the Faculty of Health Sciences Human Research Ethics Committee (HREC) (HREC REF: 909/2015).
We tested 96 isolates including 46
Among the 46
Sequence analysis of a limited number of gene products confirmed the identification of the ESBL-encoding gene
All isolates were tested for ESBL production using both phenotypic methods. Using the Sensititre method, an indeterminate result was obtained with both cefotaxime and ceftazidime for 10
The composite reference standard comprised either ESBL detection by both phenotypic methods or by a combination of one phenotypic method together with genotypic detection. Given the detection of non-ESBL genes in
The sensitivity of Vitek 2 system for detection of ESBLs as compared to the composite reference standard was 33/36 or 92% (78% – 97%) for
Extended-spectrum beta-lactamase detection by Vitek 2 compared to composite reference method in
Variable | ESBL classification according to composite reference standard |
||
---|---|---|---|
Positive | Negative | Total | |
Positive | 33 | 0 | 33 |
Negative | 3 | 10 | 13 |
ESBL, extended-spectrum beta-lactamase.
Extended-spectrum beta-lactamase detection by Vitek 2 compared to composite reference method in
Variable | ESBL classification according to composite reference standard |
||
---|---|---|---|
Positive | Negative | Total | |
Positive | 40 | 1 | 41 |
Negative | 0 | 9 | 9 |
ESBL, extended-spectrum beta-lactamase.
Detailed analysis of three
Three
The development of automated susceptibility testing systems and their subsequent introduction into routine diagnostic laboratories sparked an interest in the performance of such systems for the detection of ESBLs and a number of studies on the Vitek 2 were conducted, mostly prior to 2010.
In this study, the performance of the Vitek 2 AST-N255 card was comparable to previous studies with sensitivities of 92% (78% – 97%) and 100% (91% – 100%) and specificities of 100% (72% – 100%) and 90% (60% – 98%) for
Limitations of this study included the fact that testing was not performed in duplicate and that the commercial broth microdilution method chosen contained a limited range of antibiotic concentrations which generated many indeterminate results. Genotypic testing was limited to the commonest ESBLs, in particular to the
However, using a composite reference standard encompassing combinations of the phenotypic and genotypic methods employed in this study, no Vitek-categorised ESBL
The authors have declared that no competing interests exist.
All authors contributed equally to this work.
The authors received funding from the National Health Laboratory Service Research Trust (grant 94570) and the Federation of Infectious Diseases Societies of Southern Africa (FIDSSA)-GlaxoSmithKline Research Fellowship for Antimicrobial Resistance 2015 (no grant number provided) to carry out this research. Neither of the sponsors had any input into the design, performance or reporting of the study.
Data sharing is not applicable to this article as no new data were created or analysed in this study.
The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.
Analysis of discrepant results for extended-spectrum beta-lactamase detection.
Organism | Isolate | Resistance phenotype and susceptibility profile |
ESBL detection by disc diffusion | ESBL detection by Sensititre MIC | Dual phenotypic methods | CTX-M detected by PCR | CTX-M plus either phenotypic method | ESBL detection by composite reference standard |
---|---|---|---|---|---|---|---|---|
8 | Acquired penicillinase |
- |
+ |
- | + | + | + | |
9 | Acquired penicillinase |
- |
+ |
- | + | + | + | |
10 | Acquired penicillinase |
+ |
+ |
+ | + | + | + | |
67 | ESBL or SHV hyper-producer | - | + | - | - | - | - |
, According to CLSI criteria.
, Cefotaxime zone diameter (mm): cefotaxime + clavulanic acid zone diameter (mm) = 22:32, that is, ESBL-positive; ceftazidime zone diameter (mm): ceftazidime + clavulanic acid zone diameter (mm) = 26:27, that is, ESBL negative.
, Cefotaxime MIC (µg/mL): cefotaxime + clavulanic acid MIC (µg/mL) = 1: ≤ 0.12/4, that is, ESBL-positive; ceftazidime MIC (µg/mL): ceftazidime + clavulanic acid MIC (µg/mL) = 0.5: ≤ 0.12/4, that is, indeterminate.
, Cefotaxime MIC (µg/mL): cefotaxime + clavulanic acid MIC (µg/mL) = 0.5: ≤ 0.12/4, that is, indeterminate; ceftazidime MIC (µg/mL): ceftazidime + clavulanic acid MIC (µg/mL) = 16: 0.5/4, that is, ESBL-positive.
, Cefotaxime MIC (µg/mL): cefotaxime + clavulanic acid MIC (µg/mL) = 1: ≤ 0.12/4, that is, ESBL-positive; ceftazidime MIC (µg/mL): ceftazidime + clavulanic acid MIC (µg/mL) = 0.5: ≤ 0.12/4, that is, indeterminate.