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| Year : 2006 | Volume
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| Issue : 4 | Page : 535-539 |
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| An Accelerated Method for the Detection of Extended-Spectrum β-Lactamases in Urinary Isolates of Escherichia Coli and Klebsiella pneumoniae |
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Abdulrahman A Kader, Angamuthu Kumar, Ananth Krishna, Mohamed Nassimu Zaman
Department of Clinical Microbiology, Almana General Hospital, Al-Khobar, Saudi Arabia
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 Abstract | | |
We prospectively studied an accelerated phenotypic method by incorporating the double disk synergy test in the standard Kirby-Bauer disk diffusion susceptibility testing, to evaluate a protocol for the rapid detection of extended-spectrum B-lactamases (ESBL) in urinary isolates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). All ESBL-positive isolates were confirmed by the standard Clinical Laboratory Standards Institute (CLSI) confirmatory disk diffusion method. Between November 2004 and December 2005, a total of 6988 urine specimens were analyzed of which, 776 (11%) showed significant growth. They included E. coli in 577 cases (74%) and K. pneumoniae in 199 (25.6%). Of these, 63 E. coli (8%) and 15 K. pneumoniae (7.5%) were positive for ESBL by the accelerated and CLSI methods. Compared to the standard CLSI method, the accelerated method reduced the ESBL detection time from two days to one day. We conclude that the accelerated ESBL detection technique used by us in this study is a reliable and rapid method for detecting ESBL in urinary isolates of E. coli and K. pneumoniae. Keywords: Urinary tract infection, Escherichia coli, Klebsiella pneumoniae, Extended spectrum beta lactamase.
How to cite this article:
Kader AA, Kumar A, Krishna A, Zaman MN. An Accelerated Method for the Detection of Extended-Spectrum β-Lactamases in Urinary Isolates of Escherichia Coli and Klebsiella pneumoniae. Saudi J Kidney Dis Transpl 2006;17:535-9 |
How to cite this URL:
Kader AA, Kumar A, Krishna A, Zaman MN. An Accelerated Method for the Detection of Extended-Spectrum β-Lactamases in Urinary Isolates of Escherichia Coli and Klebsiella pneumoniae. Saudi J Kidney Dis Transpl [serial online] 2006 [cited 2022 May 18];17:535-9. Available from: https://www.sjkdt.org/text.asp?2006/17/4/535/32492 |
| Introduction | |  |
Infections by extended-spectrum betalactamase (ESBL)-producing organisms, particularly Escherichia More Details coli (E. coli) and Klebsiella pneumoniae  (K. pneumoniae, are causing significant diagnostic and therapeutic problems in afflicted patients. [1],[2] ESBLs are mutant forms of TEM-1, TEM-2 and SHV-1 enzymes coded by genes located on transferable plasmids, which can easily spread from one organism to another. [3],[4] The ESBLs are enzymes capable of inactivating a variety of CI-lactam drugs, including broad-spectrum penicillins, third-generation cephalosporins and monobactams. [5] T he ESBL-producing organisms are often multi-drug resistant, as the plasmids producing ESBLs can carry resistance to other antibiotics. [6]
The ESBL-producing bacteria are increasingly causing urinary tract infection (UTI) both in hospitalized patients and outpatients. [7] The increasing drug resistance among these bacteria has made therapy of UTI difficult and has led to greater use of expensive broad-spectrum drugs. Drug resistance of this form is often difficult to recognize using conventional antimicrobial susceptibility methods. Delay in the detection and reporting of ESBL production by gram-negative bacteria is associated with prolonged hospital stay, increased morbidity, mortality and health-care costs. [8],[9] Failure to identify ESBL-producing organisms also contributes to their uncontrolled spread. Therefore, identification of the resistant phenotypes is important, particularly in developing countries where there is excessive use of antibiotics and lack of adequate antimicrobial resistance surveillance.
ESBL-mediated resistance can be detected by several methods, including the Clinical Laboratory Standards Institute (CLSI), [10] formerly the National Committee for Clinical Laboratory Standards (NCCLS), confirmatory disk diffusion and the double-disk synergy methods. [11] The method recommended by CLSI requires a two-step approach of initially screening for ESBL production and then performing confirmatory tests on screenpositive isolates. In this study, we report our experience with an accelerated method for detection of ESBL in urinary isolates of E. coli and K. pneumoniae.
| Methods | | |
We conducted this study at the Almana General Hospitals, Al Khobar and Dammam, Saudi Arabia. They are private hospitals that have 500 beds with all specialties except transplant surgery. The study period was 14 months (November 2004 to December 2005). In this report, only samples with significant growth were studied (significant growth was defined as the presence of > 10 5 colony forming units per milliliter (cfu /ml) of urine. Isolated bacteria were identified by standard techniques [12] and the API 20E (BioMerieux, France). Antimicrobial susceptibility of the isolates was performed using Mueller-Hinton susceptibility agar plates that were inoculated with a suspension of the test strain as recommended for a standard Kirby-Bauer disk diffusion technique. [10] The following antibiotics were tested: amoxycillin (10µg), cephalexin (30µg), cefuroxime (30µg), gentamycin (10µg), amikacin (30µg), piperacillin-tazobactam (110µg) and ciprofloxacin (5µg). One of the sensitivity plates was used for ESBL detection using double-disk synergy test. [11] Disks containing ceftazidime (30µg) and cefotaxime (30µg) were placed 15 mm apart (edge to edge) from an amoxycillin (20µg)-clavulanate (10µg) (AMC) disk. Imipenem (10 µg), meropenem (10 µg), and cefepime (30µg) disks were placed on this plate. Following incubation for 18-20 hours at 35 o C, a clear extension of the zone of inhibition between ceftazidime and/or cefotaxime and AMC disk was interpreted as positive for ESBL production [Figure - 1]. The CLSI phenotypic ESBL confirmatory method, involving ceftazidime and cefotaxime with and without the inhibitor clavulanic acid, was used to confirm the presence of ESBL in each positive isolate. 10 For positive isolates, the zone diameters for cefotaxime plus clavulanic acid or ceftazidime plus clavulanic acid had to be at least 5 mm larger than the zone diameters for cefotaxime or ceftazidime alone [Figure - 2].
| Results | | |
We received and examined 6988 urine specimens between November 2004 and December 2005. Of these, 776 (11%) showed significant growth with E. Coli [577 (74%)] and K. pneumoniae [199 (25.6%)]. The initial antimicrobial susceptibility testing with the accelerated method showed that 87 isolates (11.2%) were resistant to amoxycillin, cephalexin, cefuroxime, cefotaxime and ceftazidime. Additionally, of the 776 isolates, 63 (8%) showed enhancement of the cefotaxime and ceftazidime zones towards AMC indicating ESBL presence [Figure - 1]. The 63 isolates included 48 E. coli and 15 K. pneumoniae [Table - 1]. All isolates positive by the accelerated method were also positive for ESBL by the CLSI confirmatory disk test. Of the 577 E. coli and 199 K. pneumoniae isolates, 16 (2.8%) and eight (4%) respectively, were resistant to cefotaxime and ceftazidime but negative for ESBL by both the accelerated and CLSI methods.
Carbapenems (imipenem and meropenem) were the most active antibiotics against the ESBL-producing isolates. The susceptibility data of the ESBL-producing E. coli and K. pneumoniae are summarized in [Table - 2].
| Discussion | | |
The standard CLSI method for detection of ESBL in E. coli and K. pneumoniae involves an initial screening for ESBL production and then performing confirmatory tests on screen-positive isolates. This approach may adversely affect patient care by delaying confirmation of ESBL. It is therefore more convenient to develop an accurate and rapid ESBL detection test.
We examined the performance of an accelerated protocol for the detection of ESBL by incorporating the double-disk synergy test in the standard Kirby-Bauer disk diffusion antimicrobial susceptibility testing. We placed cefotaxime and ceftazidime disks at a distance of 15 mm from AMC disk, as it has been reported to have greater sensitivity for ESBL detection than the more conventional distance of 20 to 30 mm. [13] The accelerated test results were highly concordant with that of the standard CLSI confirmation test, as they showed high sensitivity (100%) and specificity (100%). The accelerated protocol allowed the detection of ESBL-producing organisms on the same day the first line antimicrobial susceptibility results were read, thus reducing the ESBL detection time from at least two days to one day. The earlier availability of the positive results ensured appropriate antimicrobial therapy of infections caused by the ESBL-producing organisms, especially in those patients with complicated urinary tract infections not suspected to be caused by these organisms. It also allowed timely implementation of the appropriate infection control measures.
This study provides an opportunity to apply our technique to other clinical specimens where a rapid ESBL result is required. Rapid protocols for the accelerated detection of ESBLproducing organisms from blood cultures have been reported in several recent studies. [14],[15],[16]
An excellent correlation has been reported between the standard CLSI phenotypic ESBL assays and presence of ESBL-encoding genes in non- E. Coli and non-Klebsiella isolates of Enterobacteriaceae. [17],[18] However, we did not analyze the results of the accelerated procedure for other organisms in the Enterobacteriaceae family, as there are no standard CLSI recommendations for detecting ESBL in these organisms. [19]
We have now incorporated the accelerated protocol as a routine procedure on our antimicrobial susceptibility benches and are collecting data on a larger number of isolates in order to determine whether it is reliable enough to replace the standard methods for ESBL detection.
In conclusion, the protocol described in this study allowed earlier detection of ESBLproducing organisms as compared with the standard CLSI confirmation disk method.
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Correspondence Address:
Abdulrahman A Kader
Department of Clinical Microbiology, Almana General Hospital, P.O. Box 1364, Al-Khobar 31952
Saudi Arabia
 Source of Support: None, Conflict of Interest: None  | Check |
PMID: 17186689 
[Figure - 1], [Figure - 2]
[Table - 1], [Table - 2] |
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