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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 2006  |  Volume : 17  |  Issue : 4  |  Page : 535-539
An Accelerated Method for the Detection of Extended-Spectrum β-Lactamases in Urinary Isolates of Escherichia Coli and Klebsiella pneumoniae


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 2019 Nov 22];17:535-9. Available from: http://www.sjkdt.org/text.asp?2006/17/4/535/32492

   Introduction Top


Infections by extended-spectrum beta­lactamase (ESBL)-producing organisms, particularly  Escherichia More Details coli (E. coli) and Klebsiella pneumoniae Scientific Name Search  (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 Top


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 Top


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 Top


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.

 
   References Top

1.Wong-Beringer A. Therapeutic challenges associated with extended-spectrum beta­lactamase-producing Escherichia coli and Klebsiella pneumoniae. Pharmacotherapy 2001;21:583-92.  Back to cited text no. 1  [PUBMED]  
2.Livermore DM. beta-Lactamases in labora­tory and clinical resistance. Clin Microbiol Rev 1995;8:557-84.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.Sirot D. Extended-spectrum plasmid mediated beta-lactamases. J Antimicrob Chemother 1995;36 (Suppl A):19-34.  Back to cited text no. 3  [PUBMED]  
4.Tolmasky ME, Chamorro RM, Crosa JH, Marini PM. Transposon-mediated amikacin resistance in Klebsiella pneumoniae. Anti­microb Agents Chemother1988;32:1416-20.  Back to cited text no. 4    
5.Rice LE. Successful interventions for gram­negative resistance to extended-spectrum beta-lactam antibiotics. Pharmacotherapy 1999;19(8 pt 2):S120-8.  Back to cited text no. 5    
6.Steward CD, Rasheed JK, Hubert SK, et al. Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods. J Clin Microbiol 2001;39:2864-72.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Kader AA, Angamuthu K. Extended­spectrum beta-lactamases in urinray isolates of Escherichia coli, Klebsiella pneumoniae and other Gram-negative bacteria in a hospital in Eastern Province, Saudi Arabia. Saudi Med J 2005;26(6):956-9.  Back to cited text no. 7    
8.Harbarth S, Garbino J, Pugin J, Romand JA, Lew D, Pittet D. Inappropriate initial antimicrobial therapy and its effect on survival in a clinical trial of immunomodu­lating therapy for severe sepsis. Am J Med 2003;115:529-35.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Kollef MH. The importance of appropriate initial antibiotic therapy for hospital-acquired infections. Am J Med 2003;115:582-4.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Performance standards for antimicrobial susceptibility testing. Fourteenth informational supplement. Approved standard (2004) M100-S14. National Committee for Clinical Laboratory Standards, Wayne, Pa.  Back to cited text no. 10    
11.Jarlier V, Nicolas MH, Fournier G, Philippon A. Extended broad-spectrum beta lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacte­riaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis 1988;10:867-78.  Back to cited text no. 11  [PUBMED]  
12.Cowan SF, Steel KJ. Manual for identifi­cation of medical bacteria. 3 rd Ed. Cambridge: Cambridge University Press, 1993;140-3.  Back to cited text no. 12    
13.Coudron PE, Moland ES, Sanders CC. Occurrence and detection of extended­spectrum beta-lactamases in members of the family Enterobacteriaceae at a veterans medical center: seek and you may find. J Clin Microbiol 1997;35:2593-7.  Back to cited text no. 13    
14.Weinbren MJ, Borthwick MA. Rapid dete­ction of extended-spectrum beta-lactamase (ESBL)-producing organisms in blood culture. J Antimicrob Chemother 2005; 55(1):131-2.  Back to cited text no. 14    
15.Navon-Venezia S, Leavitt A, Ben-Ami R, et al. Evaluation of an accelerated protocol for detection of extended-spectrum beta­lactamase-producing gram-negative bacilli from positive blood cultures. J Clin Microbiol 2005;43(1):439-41.  Back to cited text no. 15    
16.Navon-Venezia S, Ben-Ami R, Schwaber MJ, Leavitt A, Schwartz D, Carmeli Y. Protocol for the accelerated detection of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains from blood cultures. Eur J Clin Microbiol Infect Dis 2004;2:200-2.  Back to cited text no. 16    
17.Morlote MM, Navon-Venezia S, Carmeli Y, Venkataraman L, Gold 43rd HS. Intersci Conf Antimicrob Agents Chemother abstr. 2003;C2-55.  Back to cited text no. 17    
18.Schlesinger J, Navon-Venezia S, Chlemintzki I, Hammer-Munz O, Schwaber M, Carmeli Y. Eur Congr Clin Microbiol Infect Dis Abstr 1866;2004.  Back to cited text no. 18    
19.Schwaber MJ, Raney PM, Rasheed JK, et al. Utility of NCCLS guidelines for identifying extended-spectrum beta-lactamases in non­Escherichia coli and non-Klebsiella spp. of Enterobacteriaceae. J Clin Microbiol 2004; 42:294-8.  Back to cited text no. 19    

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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
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PMID: 17186689

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