Evaluation of Methods for Ampc Β-Lactamase in Gram Negative Clinical Isolates from Tertiary Care Hospitals
Indian Journal of Medical Microbiology, (2005) 23 (2):120-124 Brief Communication
EVALUATION OF METHODS FOR AMPC β-LACTAMASE IN GRAM NEGATIVE CLINICAL ISOLATES FROM TERTIARY CARE HOSPITALS
S Singhal, T Mathur, S Khan, DJ Upadhyay, S Chugh, R Gaind, *A Rattan Abstract
The purpose of this study was to simultaneously screen for Extended-spectrum β-lactamases (ESBL) and AmpC β- lactamases in gram negative clinical isolates from four tertiary care hospitals and further to compare two detection methods three-dimensional extraction method and AmpC disk test for AmpC β-lactamases. A total of 272 isolates were screened for ESBL and AmpC ß-lactamase by modified double disk approximation method (MDDM). Synergy observed between disks of ceftazidime/cefotaxime and clavulanate were considered as ESBL producer. Isolates showing reduced susceptibility to either of the test drugs (ceftazidime or cefotaxime) and cefoxitin were considered as presumptive AmpC producers and further confirmed by three-dimensional extraction method and AmpC disk test. A total of 173 (64%) of the isolates were found to be ESBL positive and 61 (23%) showed resistant to cefoxitin. ESBL was detected in 80 (62%) isolates of E. coli and 71 (73%) of Klebsiella spp. The occurrence of AmpC β-lactamases was found to be 8% (22) of the total isolates and the two detection methods for AmpC β –lactamase showed concordant results. Screening for ESBL and AmpC can be simultaneously done by MDDM method and confirmation for AmpC ß-lactamase should be carried out routinely in tertiary care hospitals by AmpC disk test, as it is a simple and rapid procedure.
Key words: ESBL, AmpC ß –lactamases, Disk test
Extended-spectrum β-lactamases (ESBLs) and AmpC β- the standard disk diffusion breakpoint for cefoxitin (zone lactamases are of increasing clinical concern. ESBLs are most diameter <18 mm) to screen isolates and used a three– commonly produced by Klebsiella spp. and Escherichia coli dimensional extract test as a confirmatory test for isolates that but may also occur in other gram-negative bacteria. They are harbour AmpC β-lactamases. The current National Committee typically plasmid mediated, clavulanate susceptible enzymes for Clinical Laboratory Standards (NCCLS) guidelines do not that hydrolyze penicillins, expanded-spectrum cephalosporins describe any method for detection of isolates producing AmpC (cefotaxime, ceftriaxone, ceftazidime, cefepime and others) β-lactamases.5 The present study was designed to increase and aztreonam.1 AmpC class β-lactamases are awareness and demonstrate the need to detect the occurrence cephalosporinases that are poorly inhibited by clavulanic acid. of ESBLs and AmpC enzymes in clinical strains. We had They can be differentiated from other ESBLs by their ability screened the isolates by modified double disk approximation to hydrolyze cephamycins as well as other extended-spectrum method (MDDM) and selected the isolates showing reduced cephalosporins.2 AmpC- β-lactamases, demonstrated or susceptibility to ceftazidime/cefotaxime and cefoxitin or presumed to be chromosomally or plasmid mediated, have showing blunting of ceftazidime or cefotaxime zone of been described in pathogens e.g., Klebsiella pneumoniae, inhibition adjacent to cefoxitin. These isolates were further Escherichia coli, Salmonella spp., Proteus mirabilis, subjected for AmpC β-lactamases confirmation by modified Citrobacter freundii, Acinetobacter, Enterobacter spp. and three-dimensional extract test2 and AmpC disk test.6 Pseudomonas aeruginosa.3 Although reported with increasing frequency, the true rate of occurrence of AmpC β-lactamases Materials and Methods in different organisms, including members of Bacterial isolates Enterobacteriaceae, remains unknown. Coudron et al4 used A total of 272 gram negative consecutive, nonrepetitive clinical isolates from blood and urine from tertiary care *Corresponding author (email:
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Screening for ESBL and AmpC producing isolates
In order to simultaneously detect ESBL and AmpC, a modified double disk approximation method (MDDM), was devised. A 0.5 McFarland of test isolate was swabbed on Mueller Hinton Agar (Difco) plates and disk of cefotaxime (30 µg) and ceftazidime (30 µg) were placed adjacent to clavulanic acid (10 µg) and Cefoxitin (30 µg) disk at a distance of 20 mm from each other. After incubation, an enhanced zone of inhibition between any of the disks (ceftazidime / cefotaxime) and clavulanic acid were interpreted as presumptive evidence for the presence of ESBL (Fig. 1a). All ESBL positive isolates were further confirmed by determining their MICs in presence of clavulanate as per NCCLS micro broth dilution method.7 Isolates showing blunting of ceftazidime or cefotaxime zone of inhibition Figure 1a: Organism showing enhanced zone of inhibition between adjacent to cefoxitin disk (Fig. 1b) or showing reduced ceftazidime/cefotaxime and clavulanic acid disc indicating positive ESBL susceptibility to either of the above test drugs (ceftazidime or cefotaxime) and cefoxitin were considered as “screen positive” and selected for detection of AmpC β-lactamases.
Detection of AmpC β-lactamases
Modified three dimensional test
The selected isolates were studied for the presence of AmpC enzyme by modified three-dimensional extract test.2 Briefly, fresh overnight growth from Mueller Hinton agar (MHA) was transferred to a preweighed sterile micro centrifuge tube. The tube was weighed again to determine the weight of bacterial mass to obtain 10-15 mg of bacterial wet weight. The bacterial mass was suspended in peptone water and pelleted by centrifugation at 3000 rpm for 15 minutes. Crude enzyme extract was prepared by repeated freeze thawing of the bacterial pellet (approximately 10 cycles). Lawn culture of E. coli ATCC 25922 was prepared on MHA Figure 1b: Organism showing blunting of ceftazidime/cefotaxime zone plates and cefoxitin (30 µg) disk were placed on the plates. of inhibition adjacent to cefoxitin disc (presumptive AmpC producer) Linear slits (3 cm) were cut using sterile surgical blade, 3 mm away from cefoxitin disk. At the other end of the slit a small culture of E. coli ATCC 25922 was prepared on MHA plate. circular well was made and the enzyme extract was loaded. Sterile disks (6 mm) were moistened with sterile saline (20 A total of 30 to 40 µl of extract was loaded in the well at a µl) and inoculated with several colonies of test organism. The 10 µl increment. The plates were kept upright for 5 to 10 inoculated disk was than placed beside a cefoxitin disk (almost minutes until the liquid dried and were incubated at 37°C for touching) on the inoculated plate. The plates were incubated 24h. Enhanced growth of the surface organism at the point overnight at 35°C. A positive test appeared as a flattening or where the slit inserted the zone of inhibition of cefoxitin was indentation of the cefoxitin inhibition zone in the vicinity of considered a positive three-dimensional test and was the test disk. A negative test had an undistorted zone. interpretated as evidence for the presence of AmpC β- lactamases. Three different types of results were recorded. Results Isolates showing clear distortion of the zone of inhibition of Screening for ESBLs and AmpC β-lactamases cefoxitin were taken as AmpC producers. Isolates with no distortion were recorded as non-AmpC producers where as Two hundred and seventy two isolates were screened for isolates showing minimal distortion were considered as both ESBL and AmpC β-Lactamase production by modified indeterminate strains double disk approximation method (MDDM). Of this 173 AmpC Disk Test (64%) isolates were found to be ESBL positive whereas 38 were ESBL negative. Out of the total isolates 73% of All isolates subjected to three-dimensional test were also Klebsiella spp. (n=71) and 62% of E. coli (n=80) were found simultaneously checked by AmpC disk test.6 Here, a lawn to be ESBL positive (Table 1, Fig.1a).
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Of the 272 isolates, 61 showed reduced susceptibility to extraction procedure, also showed positive results by AmpC either of the test drugs (ceftazidime or cefotaxime) and disk test. Indentation indicating strong AmpC producer was cefoxitin (Table 1). Three of these isolates also showed observed in 15 isolates whereas flattening (weak AmpC) was blunting of the inhibition zone of either ceftazidime or observed in seven isolates. Three indeterminate isolates (three cefotaxime adjacent to cefoxitin disk (Fig.1b) were considered dimensional test) also showed flattening in AmpC disk test as presumptive AmpC producers and further confirmed by indicating for weak AmpC enzyme. (Fig. 3, Table 2). three-dimensional extraction method and AmpC disk test. Discussion Our results also demonstrated the co-existence phenotype β of both ESBL and AmpC in two isolates. (One each of Despite the discovery of ESBLs and AmpC -lactamases Klebsiella spp. and E. coli). at least a decade ago, there remains a low level of awareness of their importance and many clinical laboratories have Detection of AmpC β-lactamases problems in detecting ESBLs and AmpC β-lactamases. Confusion exists about the importance of these resistance Modified three dimensional test mechanisms, optimal test methods, and appropriate reporting conventions. Failure to detect these enzymes has contributed AmpC β-lactamase production was confirmed in 22 to their uncontrolled spread and sometimes to therapeutic isolates (8%) of the 272 isolates or 36% of the 61 screen failures.8 positive isolates by the three-dimensional test. A clear distortion of the zone of inhibition of cefoxitin was observed The objective of this work was to obtain some in 18 isolates whereas three isolates showed minimal experimentally based prediction on the possible emergence of distortion and were considered as indeterminate strains (Fig. ‘extended spectrum’ AmpC β-lactamases in tertiary care 2). hospitals and further to compare two phenotypic AmpC AmpC Disk Test detecting methods. Detection and reporting isolates producing AmpC β-lactamases are more difficult issues than those Of the 61 isolates 22 isolates, which were positive by associated with ESBLs. E. coli, Klebsiella pneumoniae and
Table 1: Screening for ESBLs and AmpC β-lactamases in gram negative clinical isolates
Microorganisms No. of isolates ESBL producing Screen positive resistant screened organisms (%) isolates (Suspected Amp C) E. coli 129 80 (62) 23* Klebsiella spp. 97 71(73) 18 Enterobacter spp. 8 5 (62.5) 3 Citrobacter spp. 10 8 (72.7) 2 Proteus spp. 5 4(80) 1 Pseudomonas spp. 8 3(37) 5* Acinetobacter spp. 14 2(14.2) 9 Total 272 173(64) 61 *Three isolates showed blunting of the inhibition zone of either ceftazidime or cefotaxime adjacent to cefoxitin.
Table 2: Detection of AmpC β-lactamases
Microorganisms No. of Modified three-dimensional test AmpC disk test isolates Distortion Minimal distortion No Indentation Flattening No screened (indeterminate) distortion distortion E. coli 23 7 2 14 6 3 14 Klebsiella spp. 18 4 2 12 3 3 12 Enterobacter spp. 3 2 - 1 2 - 1 Citrobacter spp. 2 1 - 1 1 - 1 Proteus spp. 1 - - 1 - - 1 Pseudomonas spp. 5 - - 5 - - 5 Acinetobacter spp. 9 4 - 5 3 1 5 Total 61 18 4 39 15 7 39
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1 3 2 1
4 5
6 5
Figure 2: Isolates showing clear distortion of the zone of inhibition (4 and 5) indicating AmpC producers, minimal distortion (1 and 2) as indeterminate and no distortion (3 and 6) as non AmpC producers by three dimensional extraction method Figure 3: AmpC disk test: isolates indicating flattening (A) and indentation (B).
Proteus mirabilis are the species in family Enterobacteriaceae AmpC harbouring isolates are largely restricted to the most commonly isolated in the clinical laboratory.9 However, hospitalized patients only.2,11 In our study AmpC β-lactamases few studies have assessed the occurrence of AmpC β- was seen mainly in Acinetobacter spp. (28.57%) followed by lactamases among these species. The NCCLS documents5 do E. coli (6.97%) and Klebsiella spp. (6.18%). The AmpC disk not indicate the screening and confirmatory test that should test was an easier, reliable and rapid method of detection of be used for the detection of AmpC β-lactamases in Klebsiella isolates that harbour AmpC β-lactamases. This suggests that pneumoniae and E. coli. AmpC disk test can be used for routine screening of the AmpC enzyme in the clinical laboratory. With the modified double disk diffusion screen test we identified 173 ESBLs and 61 isolates as possible AmpC Acknowledgement producers resistant to cefoxitin. Further investigation of this phenotypic screening method for identifying possible AmpC We appreciate the help of Dr. M. Shahid (AMU) Aligarh, Dr. Ravi (KIMS) Hubli and Dr. Arpita (BHU) Varanasi for providing the producers seems warranted, as it offers the potential to clinical isolates for this study. significantly reduce the number of isolates that would require a confirmatory test for AmpC production, such as the three- References dimensional test or AmpC disk test with cefoxitin. 1. Moland SE, Black JA, Ourada J, Reisbig MD, Hanson ND, Our initial screening also demonstrated the co-existence Thomson KS. Occurrence of newer β-lactamases in Klebsiella phenotype of both ESBL and AmpC in two isolates (one each pneumoniae isolates from 24 U.S. hospitals. Antimicrob Agents of Klebsiella spp. and E. coli). This could be because plasmid– Chemother 2002;46:3837-42. mediated AmpC enzymes have also been shown to disseminate among Enterobacteriaceae, sometimes in 2. Manchanda V, Singh NP. Occurrence and detection of AmpC β-lactamases among Gram-negative clinical isolates using a combination with ESBLs. It might be highly desirable to modified three- dimensional test at Guru Tegh Bhadur Hospital, develop an ESBL detection test that includes a substrate Delhi, India. J Antimicrob Chemother 2003;51:415-8. displaying a higher degree of resistance to such AmpC enzymes.9,10 We used cefoxitin resistance to screen isolates for 3. Bauernfeind A, Chong Y, Lee K. Plasmid- encoded AmpC beta- detecting possible AmpC β-lactamases and three dimensional lactamases: How far have we gone 10 years after the discovery? extract test and AmpC disk test for confirmation. The three Yonsei Medical Journal 1998;39:520-5. dimensional and AmpC disk test are sensitive and 22 isolates were identified by both the methods as AmpC producers. 4. Coudran PE, Moland ES, Thomson KS. Occurrence and detection of AmpC beta- lactamases among Escherichia coli, In the present study, ESBL producing isolates of Klebsiella Klebsiella pneumoniae and Proteus mirabilis isolates at a spp. (73%) and E. coli (62%) were isolated from inpatient Vetrans Medical Center. J Clin Microbial 2000;38:1791-6. units as well as from clinical samples from patients attending 5. National Committee for Clinical Laboratory Standardards. outpatient clinics. In contrast, all the AmpC harbouring Performance Standards for Antimicrobial Susceptibility Testing- organisms (8%) were found only in clinical specimens from twelfth informational supplement: Wayne, PA, USA: NCCLS; admitted patients. It has been reported that at present in India 2002;M 100-S12.
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6. Black JA, Moland ES, HossainA, Lockhart TJ, Olson LB, AmpC beta-lactamases. Emerging Infect Dis 2001;7:333-6. Thomson KS, et al. Prevalence of Plasmid-mediated AmpC β- lactamases in Klebsiella pneumoniae (KP), Klebsiella oxytoca 9. Gheldre YD, Avesani V, Berhin C, Delmee M, Glupczynski Y. (KO), Proteus mirabilis (PM), and Salmonella (S) isolates from Evaluation of oxoid combination disks for detection of extended 42 ICU and 21 non-ICU sites in the United States. Poster (C2- spectrum β-lactamases J Antimicrob Chemother 2003;52:591-7. 2034) in 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICCAC) 2003. 10. Philippon A, Arlet G, Jacoby GA. Plasmid–determined AmpC- type β-lactamases. Antimicrob Agents Chemother 2002;46:1-11. 7. National Committee for Clinical Laboratory Standardards Methods for dilution antimicrobial susceptibility tests for 11. Shahid M, Malik A, Sheeba. Multi drug resistant Pseudomonas bacteria that grow aerobically. Wayne, PA, USA: NCCLS; 2000 aeruginosa strains harboring R-plasmids and AmpC β- Standard M7-A5. lactamases isolated from hospitalized burn patients in a tertiary care hospital of North India. FEMS Microbiology Letters 8. Thomson KS. Controversies about extended–spectrum and 2003;228:181-6.
ANNOUNCEMENT XXIX National Congress of IAMM
Welcomes delegates to IAMM MICROCON 2005 - Chennai
Venue: Sri Ramachandra Medical College and Research Institute, Porur, Chennai- 600116
“Pre Conference workshops: 19.10.2005 and 20.10.2005
Venue: (i) Sankara Nethtralaya - Application of Nucleic acid based techniques(PCR) in Diagnostic Microbiology— Hands - on – Training” (ii) King Institute of Preventive Medicine - Applications of tissue culture in Toxicity and Antiviral assay of drugs— Hands-on- training. (iii) Sri Ramachandra Medical College and RI (DU) - Conventional and MolecularDiagnostic techniques in Mycology—Hands- on- Training (iv) Sri Ramachandra Medical College and RI (DU) – Bioaerosols -”Recognition, evaluation and management in health care facilities” organized by Department of Microbiology and Environmental Health Engineering, SRMC andRI, supported in part by The Fogarty International Centre, NIH , USA
Inauguration of the congress: 20.10.2005 Evening Conference Dates: 21.10.2005 - 23.10.2005
E-mail: [email protected]
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