Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 23, Num. 2, 2005, pp. 120-124

Indian Journal of Medical Microbiology, Vol. 23, No. 2, April-June, 2005, pp. 120-124

Original Article

Evaluation of Methods for AmpC Beta-Lactamase in Gram Negative Clinical Isolates from Tertiary Care Hospitals

Singhal S., Mathur T., Khan S., Upadhyay D.J., Chugh S., Gaind R., Rattan A.

New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon - 122 001
Correspondence Address: New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon - 122 001, ashok.rattan@ranbaxy.com

Code Number: mb05032

Abstract

Keywords: ESBL, AmpC β-lactamases, Disk test

Extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases are of increasing clinical concern. ESBLs are most commonly produced by Klebsiella spp. and Escherichia coli but may also occur in other gram-negative bacteria. They are typically plasmid mediated, clavulanate susceptible enzymes that hydrolyze penicillins, expanded-spectrum cephalosporins (cefotaxime, ceftriaxone, ceftazidime, cefepime and others) and aztreonam.[1] AmpC class β-lactamases are cephalosporinases that are poorly inhibited by clavulanic acid. They can be differentiated from other ESBLs by their ability to hydrolyze cephamycins as well as other extended-spectrum cephalosporins.[2] AmpC- β-lactamases, demonstrated or presumed to be chromosomally or plasmid mediated, have been described in pathogens e.g., Klebsiella pneumoniae , Escherichia coli , Salmonella spp., Proteus mirabilis, Citrobacter freundii , Acinetobacter , Enterobacter spp. and Pseudomonas aeruginosa .[3] Although reported with increasing frequency, the true rate of occurrence of AmpC β-lactamases in different organisms, including members of Enterobacteriaceae , remains unknown. Coudron et al[4] used the standard disk diffusion breakpoint for cefoxitin (zone diameter <18 mm) to screen isolates and used a three- dimensional extract test as a confirmatory test for isolates that harbour AmpC β-lactamases. The current National Committee for Clinical Laboratory Standards (NCCLS) guidelines do not describe any method for detection of isolates producing AmpC β-lactamases.[5] The present study was designed to increase awareness and demonstrate the need to detect the occurrence of ESBLs and AmpC enzymes in clinical strains. We had screened the isolates by modified double disk approximation method (MDDM) and selected the isolates showing reduced susceptibility to ceftazidime/cefotaxime and cefoxitin or showing blunting of ceftazidime or cefotaxime zone of inhibition adjacent to cefoxitin. These isolates were further subjected for AmpC β-lactamases confirmation by modified three-dimensional extract test[2] and AmpC disk test.[6]

Materials and Methods

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 plates and cefoxitin (30 µg) disk were placed on the plates. 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 circular well was made and the enzyme extract was loaded. A total of 30 to 40 µl of extract was loaded in the well at a 10 µl increment. The plates were kept upright for 5 to 10 minutes until the liquid dried and were incubated at 37°C for 24h. Enhanced growth of the surface organism at the point where the slit inserted the zone of inhibition of cefoxitin was considered a positive three-dimensional test and was interpretated as evidence for the presence of AmpC β-lactamases. Three different types of results were recorded. Isolates showing clear distortion of the zone of inhibition of cefoxitin were taken as AmpC producers. Isolates with no distortion were recorded as non-AmpC producers where as isolates showing minimal distortion were considered as indeterminate strains

AmpC Disk Test

All isolates subjected to three-dimensional test were also simultaneously checked by AmpC disk test.[6] Here, a lawn culture of E. coli ATCC 25922 was prepared on MHA plate. Sterile disks (6 mm) were moistened with sterile saline (20 µl) and inoculated with several colonies of test organism. The inoculated disk was than placed beside a cefoxitin disk (almost touching) on the inoculated plate. The plates were incubated overnight at 35°C. A positive test appeared as a flattening or indentation of the cefoxitin inhibition zone in the vicinity of the test disk. A negative test had an undistorted zone.

Results

Of the 272 isolates, 61 showed reduced susceptibility to either of the test drugs (ceftazidime or cefotaxime) and cefoxitin [Table - 1]. Three of these isolates also showed blunting of the inhibition zone of either ceftazidime or cefotaxime adjacent to cefoxitin disk [Figure 1b] were considered as presumptive AmpC producers and further confirmed by three-dimensional extraction method and AmpC disk test.

Our results also demonstrated the co-existence phenotype of both ESBL and AmpC in two isolates. (One each of Klebsiella spp. and E. coli) .

Detection of AmpC β-lactamases

Modified three dimensional test

AmpC b-lactamase production was confirmed in 22 isolates (8%) of the 272 isolates or 36% of the 61 screen positive isolates by the three-dimensional test. A clear distortion of the zone of inhibition of cefoxitin was observed in 18 isolates whereas three isolates showed minimal distortion and were considered as indeterminate strains [Figure - 2].

AmpC Disk Test

Of the 61 isolates 22 isolates, which were positive by extraction procedure, also showed positive results by AmpC disk test. Indentation indicating strong AmpC producer was observed in 15 isolates whereas flattening (weak AmpC) was observed in seven isolates. Three indeterminate isolates (three dimensional test) also showed flattening in AmpC disk test indicating for weak AmpC enzyme. [Figure - 3], [Table - 2].

Discussion

The objective of this work was to obtain some experimentally based prediction on the possible emergence of ′extended spectrum′ AmpC β-lactamases in tertiary care hospitals and further to compare two phenotypic AmpC detecting methods. Detection and reporting isolates producing AmpC β-lactamases are more difficult issues than those associated with ESBLs. E. coli , Klebsiella pneumoniae and Proteus mirabilis are the species in family Enterobacteriaceae most commonly isolated in the clinical laboratory.[9] However, few studies have assessed the occurrence of AmpC β-lactamases among these species. The NCCLS documents[5] do not indicate the screening and confirmatory test that should be used for the detection of AmpC β-lactamases in Klebsiella pneumoniae and E. coli.

With the modified double disk diffusion screen test we identified 173 ESBLs and 61 isolates as possible AmpC producers resistant to cefoxitin. Further investigation of this phenotypic screening method for identifying possible AmpC producers seems warranted, as it offers the potential to significantly reduce the number of isolates that would require a confirmatory test for AmpC production, such as the three-dimensional test or AmpC disk test with cefoxitin.

Our initial screening also demonstrated the co-existence phenotype of both ESBL and AmpC in two isolates (one each of Klebsiella spp. and E. coli ). This could be because plasmid-mediated AmpC enzymes have also been shown to disseminate among Enterobacteriaceae , sometimes in combination with ESBLs. It might be highly desirable to develop an ESBL detection test that includes a substrate displaying a higher degree of resistance to such AmpC enzymes.[9],[10] We used cefoxitin resistance to screen isolates for detecting possible AmpC β-lactamases and three dimensional extract test and AmpC disk test for confirmation. The three dimensional and AmpC disk test are sensitive and 22 isolates were identified by both the methods as AmpC producers.

In the present study, ESBL producing isolates of Klebsiella spp. (73%) and E. coli (62%) were isolated from inpatient units as well as from clinical samples from patients attending outpatient clinics. In contrast, all the AmpC harbouring organisms (8%) were found only in clinical specimens from admitted patients. It has been reported that at present in India AmpC harbouring isolates are largely restricted to the hospitalized patients only.[2],[11] In our study AmpC β-lactamases was seen mainly in Acinetobacter spp. (28.57%) followed by E. coli (6.97%) and Klebsiella spp. (6.18%). The AmpC disk test was an easier, reliable and rapid method of detection of isolates that harbour AmpC β-lactamases. This suggests that AmpC disk test can be used for routine screening of the AmpC enzyme in the clinical laboratory.

Acknowledgement

References

1.	Moland SE, Black JA, Ourada J, Reisbig MD, Hanson ND, Thomson KS. Occurrence of newer β-lactamases in Klebsiella pneumoniae isolates from 24 U.S. hospitals. Antimicrob Agents Chemother 2002;46 :3837-42.  Back to cited text no. 1
2.	Manchanda V, Singh NP. Occurrence and detection of AmpC β-lactamases among Gram-negative clinical isolates using a modified three- dimensional test at Guru Tegh Bhadur Hospital, Delhi, India. J Antimicrob Chemother 2003;51 :415-8.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.	Bauernfeind A, Chong Y, Lee K. Plasmid- encoded AmpC beta-lactamases: How far have we gone 10 years after the discovery? Yonsei Medical Journal 1998;39 :520-5.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.	Coudran PE, Moland ES, Thomson KS. Occurrence and detection of AmpC beta- lactamases among Escherichia coli , Klebsiella pneumoniae and Proteus mirabilis isolates at a Vetrans Medical Center. J Clin Microbial 2000;38 :1791-6.  Back to cited text no. 4
5.	National Committee for Clinical Laboratory Standardards. Performance Standards for Antimicrobial Susceptibility Testing-twelfth informational supplement: Wayne, PA, USA: NCCLS; 2002;M 100-S12.  Back to cited text no. 5
6.	Black JA, Moland ES, HossainA, Lockhart TJ, Olson LB, Thomson KS, et al . Prevalence of Plasmid-mediated AmpC β-lactamases in Klebsiella pneumoniae (KP), Klebsiella oxytoca (KO), Proteus mirabilis (PM) , and Salmonella (S) isolates from 42 ICU and 21 non-ICU sites in the United States. Poster (C2-2034) in 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICCAC) 2003.   Back to cited text no. 6
7.	National Committee for Clinical Laboratory Standardards Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Wayne, PA, USA: NCCLS; 2000 Standard M7-A5.  Back to cited text no. 7
8.	Thomson KS. Controversies about extended-spectrum and AmpC beta-lactamases. Emerging Infect Dis 2001;7 :333-6.   Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.	Gheldre YD, Avesani V, Berhin C, Delmee M, Glupczynski Y. Evaluation of oxoid combination disks for detection of extended spectrum β-lactamases J Antimicrob Chemother 2003;52 :591-7.  Back to cited text no. 9
10.	Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-type β-lactamases. Antimicrob Agents Chemother 2002;46 :1-11.   Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.	Shahid M, Malik A, Sheeba. Multi drug resistant Pseudomonas aeruginosa strains harboring R-plasmids and AmpC β-lactamases isolated from hospitalized burn patients in a tertiary care hospital of North India. FEMS Microbiology Letters 2003;228 :181-6.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]

Copyright 2005 - Indian Journal of Medical Microbiology

The following images related to this document are available:

Photo images