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 ~  Results
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BRIEF COMMUNICATIONS
Year : 2004  |  Volume : 22  |  Issue : 4  |  Page : 247-250
 

Detection of -Lactamases in nosocomial gram negative clinical isolates


Department of Microbiology, P. D. Hinduja National Hospital and Medical Research Center, Mahim, Mumbai - 400 016, Maharashtra, India

Date of Submission27-Oct-2003
Date of Acceptance01-Mar-2004

Correspondence Address:
Department of Microbiology, P. D. Hinduja National Hospital and Medical Research Center, Mahim, Mumbai - 400 016, Maharashtra, India

 ~ Abstract 

-lactamases represent the most common mechanism of -lactam resistance. Extended spectrum -lactamases (ESBLs) represent a major group of -lactamses currently being identified worldwide in large numbers along with inducible AmpC -lactamases and derepressed mutants. The present study was done to detect -lactamase production in clinical isolates by rearranging routine discs used in reporting susceptibility to specifically assess ESBLs, AmpC -lactamases (both inducible and hyperproducers i.e., derepressed mutants). A total of 286 clinical isolates were studied using a novel predictor disc approximation method to detect the above mechanisms of resistance with careful use and placement of antimicrobial discs. Of the 286 isolates, 151(53%) were ESBL producers of which 131(46%) were also derepressed mutants while remaining 20(7%) were plain ESBL producers. Forty (14%) were plain derepressed mutants. Inducible AmpC -lactamase production was detected in 19(7%) of the isolates. The commonest ESBL producers were E.coli and K. pneumoniae. The high incidence of -lactamase production due to multiple mechanisms in clinical isolates is alarming and urgent action needs to be taken from both a therapeutic and infection control perspective.

How to cite this article:
Rodrigues C, Joshi P, Jani S H, Alphonse M, Radhakrishnan R, Mehta A. Detection of -Lactamases in nosocomial gram negative clinical isolates. Indian J Med Microbiol 2004;22:247-50


How to cite this URL:
Rodrigues C, Joshi P, Jani S H, Alphonse M, Radhakrishnan R, Mehta A. Detection of -Lactamases in nosocomial gram negative clinical isolates. Indian J Med Microbiol [serial online] 2004 [cited 2019 Dec 16];22:247-50. Available from: http://www.ijmm.org/text.asp?2004/22/4/247/12817


B-lactamases continue to be the leading cause of resistance to -lactam antibiotics in gram negative bacteria.[1],[2] In recent years there has been an increased incidence and prevalence of extended spectrum -lactamases (ESBLs), enzymes that hydrolyze and cause resistance to oxymino-cephalosporins and aztreonam.[1],[2]
ESBLs represent a major group of -lactamases currently being identified worldwide in large numbers and are now found in a significant percentage of E.coli and K.pneumoniae strains. They have also been found in Pseudomonas.aeruginosa and other Entero-bacteriacae strains like Enterobacter, Citrobacter, Proteus, Morganella morganii, Serratia marsescens, Shigella dysenteriae, Pseudomonas aeruginosa, Burkholderia cepacia and Capnocytophaga ochracea.[1],[3]
Production of these enzymes is either chromosomally mediated or plasmid mediated.[4] Point amino acid substitution of the classical plasmid mediated -lactamases like TEM-1, TEM-2 and SHV-1 increases the spectrum of activity from earlier generation -lactams to 3rd generation cephalosporins and monobactams.[5] However, they retain their stability against cephamycins and carbapenems and are inhibited to an extent by -lactamase inhibitors.[4] Being plasmid mediated these enzymes spread fast amongst various bacteria and are important by infection control, clinical and therapeutic implication.[6] The chromosomally mediated -lactamase production is mainly through expression of AmpC gene which is either constitutive or inducible.[7]

 ~ Materials and Methods Top

All the gram negative bacteria isolated from various clinical samples, like urine, stool, sputum, pus, blood and other body fluids, of indoor patients, were selected for the study over a period of 3 months (April 2003 to June 2003). Antimicrobial susceptibility of all the isolates was performed using Kirby-Bauer disc diffusion method as per NCCLS guidelines for cefuroxime, cefotaxime, ceftriaxone, cefpodoxime, cefepime, ceftazidime, ceftazidime + clavulanic acid, imipenem, cefoxitin, aztreonam and chloramphenicol. The disc placement was designed in a novel fashion to assess ESBL and AmpC as shown in the [Figure - 1]. The ceftazidime and ceftazidime + clavulanic acid discs were kept 15-20 mm apart from each other (center to center). Imipenem, an inducer, was placed in the centre and on either side of it, at a 15-mm distance, were placed ceftazidime and cefotaxime (indicators of induction). In addition, another inducer cefoxitin was placed at 15 mm from cefotaxime (indicator). This was placed opposite to that of ceftazidime + clavulanic acid to avoid any effect of inducible -lactamase on the zone of inhibition of the latter. The remaining discs were placed as shown in the [Figure - 1].
1 - Imipenem, 2 - Cefotaxime, 3 - Cefoxitin, 4 - Ceftazidime, 5 - Ceftazidime + Clavulanic acid, 6 - Aztreonam, 7 - Ceftriaxone
An isolate was suspected to be an ESBL producer by the screening method if it had the zone sizes[8],[9] for the cephalosporins like aztreonam (30 g) 27 mm, cefotaxime (30 g) 27 mm, cefpodoxime (10 g) 21 mm, ceftazidime (30 g) 22mm and ceftriaxone (30 g) 25 mm.
We have used the following criteria for deciding an organism to be either ESBL producer, inducible AmpC producer or a derepressed mutant.[10],[11],[12]
ESBL
i) zone diameters for various 3rd generation cephalosporins as mentioned above
ii) susceptible to cefoxitin
iii) increase in zone size with addition of an inhibitor by 5 mm
Inducible AmpC
i) blunting of zone towards inducer
ii) no increase in zone size with addition of an inhibitor
iii) susceptible to cefipime
Derepressed mutants:
i) resistant to cefoxitin and cefotaxime
ii) no increase in zone size with addition of an inhibitor
Multiple mechanisms:
i) resistant to cefoxitin
ii) blunting of zone towards inducer
iii) increase in zone size with addition of an inhibitor by 5 mm

 ~ Results Top

Out of 286 isolates 151 (53%) were ESBL producers of which 131(46%) were also derepressed mutants while remaining 20(7%) were plain ESBL producers. Forty (14%) were plain derepressed mutants. Inducible AmpC -lactamase production was detected in 19(7%) of the isolates. Majority of ESBL producers and derepressed mutants were E.coli and Klebsiella spp. followed by Enterobacter and Acinetobacter spp. AmpC mediated -lactamase, both inducible as well as high level constitutive (hyperproducers), was seen in maximum number of Pseudomonas spp. Infact 18 out of 19 isolates producing inducible -lactamases were P.aeruginosa and the remaining one was Enterobacter cloacae.
All the ESBL producing as well as derepressed mutants of E.coli and Klebsiella spp. were sensitive to imipenem. Pseudomonas spp. producing plain ESBL were also sensitive to imipenem. However, eight out of 13 Pseudomonas producing both ESBL and high level AmpC and eight out of 20 producing plain high level AmpC were resistant to imipenem. Also three out of 18 producing inducible AmpC were resistant to imipenem. The percentage of different mechanisms of -lactam resistance is shown in the [Table - 1].

 ~ Discussion Top

Clinical laboratories are still not fully aware of the importance of ESBL and plasmid mediated AmpC. Although NCCLS recommendations exist they are limited to ESBL producing E.coli and Klebsiella spp. No recommendations exist for ESBL detection and reporting for other organisms or for detection of AmpC -lactamases.[3] Clinical laboratories need to develop quick screening methods to assess the mechanism of -lactam resistance in their isolates so that appropriate medication can be given. Screening methods for ESBL with recommended zone sizes should be immediately applied to suggest the presence of an ESBL. The combination with clavulanic acid bringing the susceptibility back confirms the ESBL production. In this regard cefpodoxime is the most sensitive antibiotic.[3] A standard ESBL is usually susceptible to cefoxitin and this is an adequate antimicrobial for detecting inducible resistance especially in organisms like Enterobacter, Serratia, and Citrobacter.[10],[11]
As evident from our study, resistance to cefotaxime would suggest either a derepressed AmpC or an ESBL producer. Combination with clavulanic acid bringing it back to completely susceptible level would indicate an ESBL alone. If there is an improvement with clavulanic acid, but not to the completely susceptible range, it would suggest either a derepressed AmpC + ESBL or could also suggest the presence of an ESBL with several other (non-AmpC) enzymes. Our study indicates ESBL producing E.coli and Klebsiella spp. often produce multiple -lactamases.[2]
If a strain is susceptible to cefepime and resistant to cefotaxime and cefoxitin then AmpC is a likely player. If it is cefotaxime resistant, cefoxitin sensitive and cefipime resistant or sensitive then probably it is an ESBL producer pending confirmation. High level expression of AmpC will prevent recognition of ESBL producer. This is common in strains that produce chromosomally encoded inducible AmpC -lactamases such as Enterobacter, Serratia, Pseudomonas etc. High level AmpC production has minimal effect on activity of cefipime making this drug more reliable for ESBL detection in presence of AmpC. Some authors advocate including of cefipime in differentiation of ESBL versus AmpC. However, it is important to remember that cefepime has low MIC to ESBLs, because it is a zwitterion and enters the periplasmic space efficiently. High inoculum testing generally uncovers the intrinsic activity of ESBL against cefepime.[2],[3],[6]
Ceftazidime resistance in Klebsiella pneumoniae   is a good marker of presence of ESBL.[6] Ideally the most sensitive ESBL screening agent is cefpodoxime for Klebsiella spp. and E.coli.[3] Today it is commonplace for Klebsiella spp. to produce 3 to 6 types of -lactamases and these changes in bacterial pathogens necessitate new and modified tests to provide accurate and clinically relevant susceptibility reports.[2],[3],[6]
In our study it is apparent that various different mechanisms exist for production of multiple -lactamases especially in high pressure units, such as intensive care units (ICU), where newer -lactams are being routinely prescribed. The carbapenems still retain adequate activity for almost all Klebsiella spp. and E.coli and for some Pseudomonas spp. (70%). However, we need to keep in mind that the carbapenems are antimicrobials that are usually kept in reserve. The marked increase in -lactamase production, including the high level constitutive producers (derepressed mutants) with ESBL, leave us with few alternatives in combating serious infections. Good infection control practice and careful introspection, while prescribing -lactam drugs with a background of the ICU flora are necessary for good antimicrobial stewardship in hospitals. 

 ~ References Top

1.Bradford PA. Extended spectrum -lactamases in the 21st century: Characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001;14(4):933-951.  Back to cited text no. 1    
2.Rice LB, Bonomo RA. -lactamases: which ones are clinically important? Drug Resistance Update 2000;3:178-189.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.Thomson KS. Controversies about Extended spectrum and AmpC beta lactamases. Emerging Infectious Diseases 2001;7(2): 333-336.  Back to cited text no. 3    
4.Morlote MM. Extended spectrum -lactamases. Infectious Diseases Case Conference Oct 2001.  Back to cited text no. 4    
5.Steward CD, Rasheed JK, Hubert SK, Biddle JW, Raney PM, Anderson GJ, Williams PP, Brittain KL, Oliver A, McGowan JE Jr, Tenover FC. Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using National Committee for Clinical Laboratories Standards Extended spectrum -lactamase detection methods. J Clin Microbiol 2001; 39(8):2864-2872.  Back to cited text no. 5    
6.Beringer AW. Therapeutic challenges associated with Extended spectrum -lactamase producing Eschcerichia.coli and Klebsiella pneumoniae. Pharmacotherapy 2001;21(5):583-592.  Back to cited text no. 6    
7.Livermore DM. -lactamases in laboratory and clinical resistance. Clin Microbiol Rev 1995;8(4):557-584.  Back to cited text no. 7    
8.Blondeau JM. Extended spectrum -lactamases. Seminars in Respiratory Infections 2001;16(3):169-176.  Back to cited text no. 8    
9.Guidelines on susceptibility of antibiotic resistant Enterobacteriaceae due to extended spectrum -lactamases (ESBL). Canadian External Quality Assurance Advisory Groups on Antimicrobial Resistance (CEQA - AGAR) and Bureau of Microbiology; Health Canada, December 1999.  Back to cited text no. 9    
10.Moritz VA, Carson PBD. Cefoxitin sensitivity as a marker for inducible beta lactamases. J Med Microbiol 1986;21:203-207.  Back to cited text no. 10    
11.Revathi G, Singh S. Detection of expanded spectrum cephalosporin resistance due to inducible lactamases in hospital isolates. Indian J Med Microbiol 1997;15(3):113-115.  Back to cited text no. 11    
12.National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Twelfth informational supplement. M100 - S12 NCCLS, 2002. Wayne P. A.  Back to cited text no. 12    
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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04