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Original Research: Detection of Extended-Spectrum β-lactamases in AmpC β-lactamase-Producing Nosocomial Gram-negative Clinical Isolates from a Tertiary Care Hospital in Delhi

Author(s): Rajini E, Sherwal BL, Anuradha

Vol. 4, No. 6 (2008-01 - 2008-02)

Rajini E, Sherwal BL, Anuradha

ISSN: 0973-516X

Dr Ekadashi Rajni, MD, (Senior Resident), Dr Anuradha, (Junior Resident), and Dr B L Sherwal, MD, (Professor) are from the Department of Microbiology, Lady Hardinge Medical College, Delhi.

Correspondence: Dr Ekadashi Rajni,
D-22,Rose Apartment,Sector-14 extension, Rohini, Delhi-110085
[Email address:ravajni (at)]


Enterobacteriaceae producing both AmpC β lactamases and ESBLs have been increasingly reported worldwide. The purpose of this study was to simultaneously screen for ESBL and AmpC v lactamases in nosocomial gram-negative clinical isolates from a tertiary care hospital in Delhi. A total of 282 isolates from various clinical samples collected during January to June 2007 were included in the study. ESBL production was noticed in 160 (56.7%) isolates with maximal incidence in E. coli (72%) followed by Klebsiella pneumoniae (38.4%). 165 (58.5%) strains were found to harbor AmpC enzyme, the maximal incidence being in E.coli (70%) followed by Klebsiella pneumoniae (56.7%). Co- production of ESBL and AmpC β lactamases was seen in 113 (40%) isolates. Amongst the AmpC producers, resistance to cefepime and imipenem was seen in only 2.4% and 3% strains respectively. The high incidence of β lactamase-production due to multiple mechanisms is alarming and requires urgent action from both a therapeutic and infection control perspective.

Key Words: ESBL, AmpC β lactamase, gram-negative isolates, co-production of the two β-lactamases.


Extended-spectrum β-lactamases are enzymes that mediate resistance to extended-spectrum cephalosporins (ESCs) and the monobactam, Aztreonam1, 2. They have been well characterized in a significant percentage of gram negative bacilli. ESBLs are predominantly derivatives of TEM and SHV enzymes and are inhibited by clavulanic acid.1-3

Though classically defined, confirmation of ESBL production by clavulanic acid inhibition can be difficult in some strains. This is because, not only the activity of the β-lactamases varies with different substrates, but also because organisms may contain additional resistance mechanisms which can mask the presence of ESBL activity. These additional mechanisms include AmpC type of enzymes, porin changes or TEM and SHV enzymes that are no longer inhibited by clavulanic acid (CA) due to mutations in coding sequences4-7.

AmpC class of β-lactamases (cephalosporinases) is poorly inhibited by clavulanic acid8. They can be differentiated from other ESBLs by their ability to hydrolyze cephamycins as well as other ESCs. More recently, and ominously, the class C enzymes have found their way onto plasmids, by means of which they have been disseminated among bacterial strains.1-3,8,9.

AmpC-producing organisms act as hidden reservoirs for ESBLs.8-12 Their coexistence further complicates the picture because high level expression of AmpC β-lactamase may mask the recognition of ESBLs10,13,14. Such isolates when tested by CA inhibition tests, are induced to produce high levels of AmpC enzymes which may antagonize the synergy arising from inhibition of ESBLs.15 In such a situation, much better inhibition is achieved if tazobactam is used instead of Amoxy-clav or if Cefipime is used as an indicator cephalosporin (since high level AmpC production does not antagonize cefepime activity).12,13

The present study was designed to enable us to detect both ESBL and AmpC β-lactamase production in gram-negative isolates from inpatients in a tertiary care hospital in Delhi in a cost effective manner.

Materials & Methods

A total of 282 consecutive, non-repetitive, gram-negative clinical isolates over a period of 6 months (January –June2007) from a variety of clinical samples as pus, wound swab, body fluids, sputum, high vaginal swabs and blood of indoor patients processed at Lady Hardinge Medical College (Delhi) were included in the study. The isolates were subjected to antimicrobial susceptibility testing to 15 different antimicrobial agents (Ceftazidime Ca, 30μg; Cefotaxime Ce, 30μg; Ceftriaxone Ci, 30μg; Cefipime Cpm, 30μg; Cefoxitin Cn 30μg; Cotrimoxazole Co, 1.25/23.75μg; Amikacin AK, 30μg; Gentamicin G, 10μg; Netilmycin Nt, 30μg; Ciprofloxacin Cf, 5μg; Pipercillin Pc, 100μg; Pipercillin- Tazobactam PTZ, 100/10μg; Augmentin Ac, 20/10μg; Imipenem I, 10μg) by standard disc-diffusion method using commercially available discs (HIMEDIA) as per CLSI guidelines16. Quality control was achieved using a standard strain of E. coli ATCC 25922.

In order to simultaneously screen for ESBL and AmpC β-lactamases, the placement of discs used was as shown in Fig 1. Ca, Ci and Ce discs were placed on either side of Ac disc 15-20 mm apart (centre to centre) from each other. Cpm and PTZ discs were also placed 15-20 mm apart (centre to centre) from each other. Cn disc (an inducer), was placed at 15mm from Ce disc (an indicator) on the side opposite to that of Ac (to avoid any effect of inducible β-lactamase on the zone of inhibition of the latter). A 0.5 Mc Farland of test isolate was swabbed on Mueller-Hinton agar plate and discs placed as described above. After incubation, an organism was considered as an ESBL producer if there was an enhancement of the zone of inhibition between any of the discs. Isolates showing blunting of Ce zone of inhibition adjacent to Cn disc or showing a cefoxitin zone of inhibition <18mm were considered as screen positive and further confirmed for AmpC enzyme production by AmpC disc test.

Amp C DISK TEST: Here a lawn culture of E. coli ATCC 25922 was prepared on MHA plate. Sterile disks (6mm) were moistened with sterile saline (20μl) and inoculated with several colonies of test organisms. The inoculated disk was then placed beside a cefoxitin disk, almost touching it, on the inoculated plate. The plates were then incubated overnight at 35°C. A positive test appeared as a flattening / indentation of the cefoxitin zone of inhibition in the vicinity of the test disk. A negative test had an undistorted zone.18


Of the 282 non-repeat gram-negative isolates that were included in the study, ESBL production was noticed in 161(57%) isolates with maximal incidence in E.coli (72%; n=117) followed by Klebsiella pneumoniae, (38%; n=40). Eighty percent (n=226) showed either blunting of Cefotaxime zone of inhibition adjacent to Cn disc or showed Cn zone of inhibition 18mm and were considered as screen positive for AmpC production. Of these, 165 (58.5%) strains were confirmed to harbor AmpC enzyme by AmpC disk test. Maximal incidence of AmpC production was found among E. coli (70%; n=102) followed by Klebsiella pneumoniae, (56.7%; n=59). Of the 163 AmpC-β lactamase producers, resistance to cefipime and imipenem was noticed in a mere 2.4% (n=4) and 3% (n=5) strains respectively. However, 73.6 % (n=120) strains showed resistance to ciprofloxacin.

Fig 1. Scheme of disc placement to assess ESBL and AmpC production

Co-production of AmpC β-lactamases and ESBLs was seen in 40% (113 isolates), the maximal incidence of co-production of several varieties of β-lactamases seen again in E.coli (44.4%). Of the 113 strains which produced both AmpC β-lactamases and ESBLs, Ca-Ac combination could detect ESBL production in only 5 strains, while in the remaining 108 strains, ESBL production could be detected only using CPM-PTZ combination, thereby suggesting this combination to be more sensitive for detecting ESBL production in the presence of AmpC β-lactamases. However, in one strain of E.coli exhibiting coexistence of these enzymes, ESBL phenotype was detected using Ca-Ac and not Cpm-PTZ.

Table 1. ESBL and AmpC production in various gram negative isolates

No .
No. of
ESBL producers Screen positive
isolates for
AmpC production
Both AmpC
Both Total
N (%)
1. E.coli 162 34 116 33 117 (72%) 138 (85%) 102 (70%) 72 (44.4%)
2. Klebsiella spp. 104 10 40 10 40 (38.4%) 83 (79.8) 59 (56.7%) 38 (36.5%)
3. Pseudomonas spp. 6 1 1 0 2 (33.6%) 2 (33.3%) 1 (16.6%) 1 (16.6%)
4. Acinetobacter spp. 10 0 2 0 2 (20%) 3 (30%) 3 (30%) 2 (20%)
  Total 282 45 159 43 161 (57%) 226 (80%) 165 (58.5%) 113 (40%)

Of the 6 strains of Pseudomonas aeruginosa studied, ESBL and AmpC β-lactamase production was seen in two and one isolate, respectively, with one showing co-production of both. Ten strains of Acinetobacter spp were also included in the study. ESBL and AmpC production was seen in 20% and 30% strains respectively with two (20%) strains showing co-production of both.


Existence of multiplicity of resistance mechanisms in gram-negative isolates remains a grey area for most of the clinical microbiologists. Despite the discovery of such organisms at least a decade ago, clinical laboratories are still not fully aware of their importance. Confusion exists about the optimal test methods and appropriate reporting protocols12. Although CLSI recommendations exist they are limited to ESBL-producing E. coli and Klebsiella species. No recommendations exist for ESBL detection and reporting for other organisms and for detection of AmpC beta lactamases.16 Failure to detect these organisms has contributed to their uncontrolled spread and the consequent clinical failures.4,12 Distinguishing between the two β-lactamases is of considerable therapeutic interest to a clinical microbiologist. The objective of the present study was to obtain some lab-based data to highlight the importance of the presence of multiple resistance mechanisms in gram-negative isolates and to underline the need for development of clear-cut strategies for their detection.

The incidence of ESBL in major hospitals of India has been reported to be as high as 60%-80%. Worldwide prevalence has been non-uniform. Some hospitals in the US appear to have no ESBL problem, while in other hospitals as many as 40% of Klebsiella pneumoniae isolates have been reported to be ceftazidime resistant because of ESBL production2. In Taiwan, Yan et al19 have reported 94% Klebsiella spp as ESBL producers.

Of the 282 strains included in our study 57% showed ESBL production, with the highest incidence in E.coli (72%) followed by Klebsiella spp (38%). Our results are in concordance with some other studies24. However, lower percentages were reported from Chennai25 (20%) and Hyderabad26 (19.8%).

For detection of AmpC class of β-lactamases, no satisfactory technique has been established till date, although various researchers have tried the 3-D test with different modifications. This technique, first described by Thomson et al27, has its own limitations, despite being increasingly sensitive. In our study, we used an AmpC disk test which is an easier, reliable and rapid method of detection of isolates that harbour AmpC β-lactamase. This suggests that AmpC disk test can be used for routine screening of the AmpC enzymes in clinical laboratory18,28.

In 2003, 20.7% AmpC producers were found among gram-negative isolates in Guru Tegh Bahadur Hospital, Delhi8. In the same year, Subha et al found AmpC β-lactamase production in 24.1% of Klebsiella spp and 37.% of E.coli in Chennai29. Around the same time, Shahid et al30 reported 20 P. aeruginosa isolates as producing AmpC beta lactamases in Aligarh30, while in Karnataka, 3.3% of E.coli and 2.2% Klebsiella pneumoniae isolates were found to harbour AmpC enzymes31. In 2005, data from Chennai32 revealed AmpC production in 20.8% Klebsiella spp and 16.6% E.coli. The data generated in our hospital reveal a relatively higher percentage of gram negative isolates as producing these enzymes (70% E.coli and 57% Klebsiella spp). Our findings are consistent with those of Woodford et al22 in UK and Ireland, 2007 and Dunne et al33 from Missouri, (85.5%). This recent increase in AmpC producing isolates may be indicative of the ominous trend of more and more isolates acquiring resistance mechanisms rendering the antimicrobial armamarium ineffective.

Our results show that Klebsiella spp. harbour AmpC enzymes less frequently than E. coli does.(57% vs 70%). Yan et al19 in 2006 have reported similar findings from Taiwan (14.5 vs 43.6%). The higher ncidence of AmpC β-lactamases in E.coli may reflect two modes of production: hyper production of chromosome mediated AmpC and plasmid mediated AmpC beta-lactamases.

In our study while cefoxitin-resistance was evident in 80% isolates, only 58.5% isolates were found to harbour AmpC enzymes i.e. 28% of isolates with reduced susceptibility to cefoxitin did not harbour AmpC enzymes. In these strains, cefoxitin resistance can be explained by loss of porins. Hernandez et al7 demonstrated that interruption of a porin gene by insertion sequences is a common type of mutation that causes the loss of porin expression and increased cefoxitin resistance. Ananthan et al32 in Chennai got similar results in 2005 where resistance to cefoxitin was mediated by both AmpC β-lactamase production and loss of OMP.

Among the 165 AmpC producers only 2.4% and 3% strains, respectively, exhibited resistance to Cefipime and Imipenem, thereby reiterating the continued efficacy of carbapenems and fourth generation cephalosporins as the first line agents for treatment of nosocomial infections caused by Enterobacteriaceae producing ESBL and AmpC beta lactamases. Similar data has been published in the MYSTIC Program in Europe and the US (1997-2004) and other studies which claim that worldwide 99.9% of Enterobacteriaceae remain susceptible to carbapenems2,11.

The coexistence of Amp C and ESBL was found in 44.4% E. coli and 36.5% Klebsiella spp in our study. A similar frequency (35%) was reported by Yan et al from Taiwan in 2004 in Klebsiella spp10. Wang QT et al, however, reported a lesser prevalence of AmpC enzymes among 2% and 17.1% ESBL producing E.coli and Klebsiella pneumoniae isolates respectively34. This could be because plasmid-mediated AmpC enzymes have also been shown to disseminate among Enterobacteriaceae, sometimes in combination with ESBLs. In such situations, it is desirable to develop an ESBL detection test that includes a substrate displaying a higher degree of resistance to such AmpC enzymes as cefepime. Also tazobactam is preferable over augmentin. In the present study, additional 82 and 30 strains, respectively, of E.coli and Klebsiella spp. were detected using cefepime-PTZ combination than with using ceftazidime-augmentin. There was only one strain of E.coli whose ESBL phenotype was detected by Ca-Ac but not by Cpm-PTZ. This could be due to the variation in substrate profile of the various ESBLs.

The placement of discs as used in the present study facilitates the screening of gram negative isolates for co-production of several enzymes in a single step. Given the fact that many clinical laboratories are often short-staffed and overworked, this new template can help fill an existing gap in clinical microbiology. For laboratories attached to tertiary care centers, handling a large number of samples, it allows continuous surveillance of the prevalence and evolution of these enzymes, thereby reducing the escalation of antibiotic resistance through better infection control.


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