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Year : 2014  |  Volume : 57  |  Issue : 2  |  Page : 255-258
Characterization of plasmid mediated AmpC producing Escherichia coli clinical isolates from a tertiary care hospital in South India

1 Department of Medicine, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India
2 Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India

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Date of Web Publication19-Jun-2014


Context: Plasmid mediated AmpC (pAmpC) β-lactamase producing Escherichia coli are an emerging problem worldwide as they are now exhibiting resistance to multiple classes of antibiotics and are a major cause of therapeutic failure. Aims: The aim of this study was to characterize pAmpC β-lactamase producing extraintestinal E. coli, their phylogenetic distribution, resistance pattern, treatment options, and impact on patient's clinical outcome. Settings and Design: This descriptive study was carried out in a multi-specialty tertiary care hospital. Materials and Methods: A total of 300 clinically significant, non-repeat isolates were studied. AmpC disk test was used for phenotypic AmpC-β-lactamase detection. Molecular types of pAmpC were determined by a multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Metallo-beta-lactamase (MBL) detection was done by E test. Antibiogram, treatment, and clinical outcome were collected in a structured proforma. Results: Although 95 isolates (32%) were phenotypically positive for AmpC, PCR detected CIT type of AmpC gene in only 37 isolates. Majority of strains were from phylogroup A (85%) and B1 (58%) which are considered as commensal groups. Co-production of ESBL's was observed in 33 strains and 5 strains were found to be MBL producers. Most widely prescribed antibiotics were 3 rd generation cephalosporins (30%), carbapenems (19%) and aminoglycosides (16%). Conclusions: Plasmid mediated AmpC producing isolates were found to exhibit a high degree of drug resistance, and they mainly belonged to commensal strains possibly due to misuse of antibiotics. Proper antibiotic policy is required to limit the spread of pAmpC producers or else it will lead to a therapeutic dead end in the near future.

Keywords: Clinical outcome, multidrug-resistance, multiplex polymerase chain reaction, phylogenetic group, plasmid mediated AmpC β-lactamases

How to cite this article:
Chakraborty A, Adhikari P, Shenoy S, Saralaya V. Characterization of plasmid mediated AmpC producing Escherichia coli clinical isolates from a tertiary care hospital in South India. Indian J Pathol Microbiol 2014;57:255-8

How to cite this URL:
Chakraborty A, Adhikari P, Shenoy S, Saralaya V. Characterization of plasmid mediated AmpC producing Escherichia coli clinical isolates from a tertiary care hospital in South India. Indian J Pathol Microbiol [serial online] 2014 [cited 2023 Mar 31];57:255-8. Available from:

   Introduction Top

0 Escherichia More Details coli naturally produce a very low level of chromosome encoded AmpC-β-lactamase enzyme. [1] However, hyper-production of this enzyme can cause resistance to penicillins, first and second generation cephalosporins, cephamycins and to oxyimino cephalosporins. Genetic analysis of hyper-producing strains revealed that mutation in the AmpC promoter, attenuator regions and also acquisition of plasmid carried AmpC genes are responsible for AmpC over production. [2] Although plasmid mediated AmpC-β-lactamases (pAmpC) have been known since 1988, a large number of infectious disease personnel remain unaware of their clinical importance. [3] All plasmid carried AmpC genes have significant clinical relevance. Several studies have shown high rates of treatment failure due to pAmpC producers. [2],[4] The objective of the present study was to characterize pAmpC β-lactamases expressed by clinical E. coli isolates, their phylogenetic distribution, resistance pattern, and impact on patient's clinical outcome.

   Materials and Methods Top

Clinical isolates

The study was conducted on 300 non-repeat extraintestinal E. coli isolates from hospitalized patients during the period from August 2010 to January 2012. The strains were isolated from patients with urinary tract infection (n = 159), sepsis (n = 77), wound infection (n = 40), pneumonia (n = 19), intra vascular device infection (n = 3), and meningitis (n = 2). After obtaining permission from the Institutional Ethical Committee the clinical data from the patient's records were collected in a structured proforma.

Antibiotic susceptibility testing

Antibiotic susceptibility testing was performed by the modified Kirby-Bauer disk diffusion method, and interpretation was done according to 2010 Clinical and Laboratory Standards Institute (CLSI) guidelines. [5]

Phenotypic AmpC activity testing

Isolates were tested for AmpC enzyme production by AmpC disk test. [3] Briefly, a suspension of ATCC E. coli 25922 standardized to 0.5 McFarland was inoculated on the surface of Mueller-Hinton agar plate. A 30 μg cefoxitin disk was placed on the inoculated surface of the agar. A sterile plain disc containing tris-ethylenediaminetetraacetic acid (EDTA) inoculated with several colonies of the test isolate was placed beside the cefoxitin disc almost touching it, with inoculate in contact with the agar surface. The plates were incubated overnight at 35°C aerobically. A positive test was indicated by flattening or indentation of cefoxitin inhibitory zone in the vicinity of the test disc. A negative test had an undistorted zone. Isolates which were AmpC producers were further tested for extended-spectrum beta-lactamase (ESBL) carbapenemases and metallo-beta-lactamase (MBL) activities, as described elsewhere. [6]

Molecular detection of plasmid mediated AmpC β-lactamase genes

Isolates which were resistance to cefoxitin and positive by AmpC disk test were tested for the presence of plasmid carried AmpC-β-lactamase genes. Briefly, template DNA was obtained by boiling-lysis method. Following genes normally MOX, CIT, DHA, ACC, EBC, and FOX were amplified by using the oligonucleotide primers as described earlier. [7] The polymerase chain reaction (PCR) was performed in a final reaction volume of 50 μl containing 750 Mm tris-HCl, 50 mM KCl, 1.5 mM MgCl 2 0.2 mM each dNTP, 0.6 μM primers MOXM, CITM, DHAM, 0.5 μM primers ACCM, EBCM, and 0.4 μM primers FOX; 1.25 U of Taq DNA polymerase and 2 μl template DNA. An Eppendorf thermocycler was used for amplification. The program for amplification included a step of initial denaturation at 94°C for 3 min followed by 25 cycles of 94°C for 30 s, 64°C for 30 s and 72°C for 1 min and a final extension step at 72°C for 7 min. The PCR products were loaded in 2% wt/vol agarose gel prepared in tris-borate-EDTA buffer at 120 V for 1 h and detected by ethidium bromide staining after electrophoresis.

Phylogenetic grouping of the AmpC producing isolates

Isolates were assigned to one of the four main phylogenetic groups of E. coli (A, B1, B2, and D) by using the triplex PCR as described by Clermont et al. [8] Briefly, template DNA was obtained by boiling-lysis method. Following genes chuA, yjaA, and TSPE4.C2 were amplified by using the oligonucleotide primers as described earlier. [8] The PCR was performed in a final reaction volume of 50 μl containing 750 Mm tris-HCl, 200 mM (NH 4 ) 2 SO 4 , 2.5 mM MgCl 2 0.2 mM each dNTP, 0.4 μM of each primers, 1 U of Taq DNA polymerase and 5 μl template DNA. An Eppendorf thermocycler was used for amplification. The program for amplification included a step of initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, and a final extension step at 72°C for 7 min.

The PCR products were loaded in 2% w/vol agarose gel prepared in tris-borate-EDTA buffer at 120 V for 1 h and detected by ethidium bromide staining after electrophoresis.

   Results Top

A total of 300 extraintestinal E. coli isolates were screened for the AmpC phenotype by AmpC disk test. Of these, 95 isolates were found to be AmpC producers by disk test. Among the 95 isolates, CIT type of plasmid was found in 37 isolates by PCR method [Figure 1]. Blood isolates were carrying maximum number (45%) of pAmpC compared with other isolates. Twenty seven percent (10/37) pAmpC producers were isolated from hospital acquired infection and 73% (27/37) were from community acquired infections. Phylogenetic distribution of the pAmpC isolates showed 85% of the isolates were from Group A followed by Group B (58%) [[Table 1] and [Figure 2]. Among the pAmpC producing isolates, 89% were ESBL co-producers, whereas only 11% were pure pAmpC producers. pAmpC producing isolates also produced carbepenemases and MBL enzymes [Table 2]. The occurrence of antimicrobial resistance in the pAmpC producing isolates was as follows, piperacillin/tazobactam 84% (31/37), cefoperazone/sulbactam 46% (17/37), amikacin 40% (15/37), gentamcin 73% (27/37), ciprofloxacin 87% (32/37), co-trimoxazole 60% (22/37), norfloxacin 51% (19/37), ampicillin/sulbactam 54% (20/37), imipenem 16% (6/37), meropenem 19% (7/37), and etrapenem 5% (2/37). However, with appropriate antimicrobial treatment 35% patients infected with pAmpC producing isolates clinically improved. For treatment of patients infected with AmpC producing isolates common prescribed group of antibiotics were carbapenemase aminoglycoside and 3 rd generation cephalosporins.
Figure 1: Image of polymerase chain reaction (PCR) for pAmpC gene detection in our isolates: 2% Agarose gel showing product of PCR amplification. Lane 1: 100 bp molecular size standard DNA ladder. Lane 2: negative water control. Lane 3: positive control with 462 bp CIT gene. Lanes 4 and 5:  Escherichia coli Scientific Name Search olates) with 462 bp CIT gene

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Figure 2: Triplex polymerase chain reaction results of phylogenetic grouping of extraintestinal pathogenic Escherichia coli isolates: Phylogenetic Group A [(chu A-, yjaA-, TspE4.C2-) and (yjaA+, chu A-, TspE4.C2-)]; Group B1 [chu A-, yjaA-, TspE4.C2+]; Group B2 [(chuA+, yjaA+, TspE4.C2-) and (chuA+, yjaA+, TspE4.C2+)]; and Group D [(chuA+, yjaA-,TspE4.C2-) and (chuA+, yjaA-, TspE4.C2+)]

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Table 1: Differentiation between pAmpC producing isolates with non-pAmpC producing isolates

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Table 2: Distribution of β-lactamase producers among pAmpC positive and negative isolates

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   Discussion Top

Plasmid mediated AmpC-β-lactamases belonging to Ambler class C are a new threat worldwide as they mediate resistance to a broad spectrum of antibiotics. In recent years, pAmpC is increasingly identified in E. coli.

Detection of AmpC β-lactamases in E. coli is a challenge to microbiological laboratories as there are no CLSI guidelines and molecular detection is not possible in all laboratories. However, proper recognition of pAmpC producing E. coli strains is important for clinical management and epidemiological surveillance.

In our study population, 32% of the isolates were found to be AmpC producers by disk test, whereas only12% isolates were positive for pAmpC. In recent years, incidence of pAmpC positive E. coli has been reported from various part of the country. [9],[10] Almost all types of pAmpC (CIT, DHA, MOX, ACC, and CMY) are common in India. In our study, we found that only the CIT family of pAmpC was common among our isolates.

In this study, we found maximum number of isolates were from blood and urine, which is similar to other study findings. [10],[11]

Several studies have reported that pAmpC producers are isolated mainly from patients with prolonged hospitalization, but certain recent studies have reported that pAmpC producers were also isolated from long term care facilities and outpatient clinics, which indicate the presence of pAmpC in the community. [2],[9],[12] In the present study, we also isolated pAmpC producers from both hospital- and community-acquired infected patients.

Studies have shown that almost all chromosomal AmpC overexpressing isolates belonged to commensal phylogroups A or B1, which are less virulent then the virulent B2 and D phylogroups. [13] However, not much data exists regarding occurrence of AmpC producing strains and their phylogenetic distribution from India. In our study, we found that majority (approximately 65%) of pAmpC isolates belonged to the commensal phylogroups (Group A and B1). This indicates that virulent strains were carrying less pAmpC genes as compared with commensal strains.

In this study, we found the occurrence of a high degree of multidrug-resistance among the pAmpC producers. Although, AmpC enzyme is inhibited by tazobactam, a high resistance rate was found in pipracillin/tazobactam combination among our isolates, which is similar to other studies. [9],[10] This may be due to the presence of other β-lactamase enzymes of the isolates as most of the isolates were ESBL co-producers. About 22% of pAmpC producers were carbapenemase producers and 14% were also MBL producers.

For E. coli infections frequently used group of antibiotics are fluoroquinolones, cephalosporins and β lactam+ β-lactamase inhibitor combinations. However, AmpC producing strains are often resistant to multiple agents making selection of an effective antibiotic difficult. Beta lactam+ β-lactamase inhibitor combinations and most cephalosporins and penicillins should be avoided because of either in vitro resistance or the potential for AmpC induction. Selection of high enzyme level mutants has been documented with poor clinical outcomes for ceftazidime and cefotaxime. [4] Cefepime can be a drug of choice for AmpC producers as cefepime is a poor inducer of AmpC β-lactamase, rapidly penetrates through the outer cell membrane, and is less hydrolysed by the enzyme. [2] However, in our study, none of the patients received cefepime as treatment. We recommend that cefepime should be included in hospital antibiotic policy, carbapenems can be used for the treatment of AmpC producers, but carbapenem resistance can rise in isolates due to outer membrane porin loss (reduced influx or efflux pump activation).

Outcome of our study indicates that maximum number of patients improved with proper antibiotic treatment whereas one in five patients expired due to infection caused by multi-drug resistant pAmpC producing isolates.

Our study had certain limitations, in that we studied only pAmpC producing isolates. However, other mechanisms for hyper production of AmpC enzyme such as due to chromosomal mutations were not studied and it is difficult to demonstrate attributable mortality solely to infection without proper study design and/or autopsy to provide evidence as those patients had some underlying conditions.

   Conclusion Top

Our finding suggests that spread of E. coli pAmpC is not solely because of hospital sources, but also due to the emergence of pAmpC in patient's commensal strains, possibly through antimicrobial selective pressure. Hence, knowledge regarding the source, type of pAmpC, antibiogram, treatment options, and patient's clinical outcome is required to make proper antibiotic and hospital policy so as to reduce spread of pAmpC, whereas in the absence of novel agents in the near future, the spread of pAmpC producers may lead to therapeutic dead ends.

   Acknowledgment Top

We are grateful to Manipal University, Manipal, India and Association of Physicians, Karnataka, for providing infrastructure and financial support respectively, to conduct the study.

   References Top

1.Caroff N, Espaze E, Gautreau D, Richet H, Reynaud A. Analysis of the effects of -42 and -32 ampC promoter mutations in clinical isolates of Escherichia coli hyperproducing ampC. J Antimicrob Chemother 2000;45:783-8.  Back to cited text no. 1
2.Jacoby GA. AmpC beta-lactamases. Clin Microbiol Rev 2009;22:161-82.  Back to cited text no. 2
3.Black JA, Moland ES, Thomson KS. AmpC disk test for detection of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J Clin Microbiol 2005;43:3110-3.  Back to cited text no. 3
4.Pai H, Kang CI, Byeon JH, Lee KD, Park WB, Kim HB, et al. Epidemiology and clinical features of bloodstream infections caused by AmpC-type-beta-lactamase-producing Klebsiella pneumoniae. Antimicrob Agents Chemother 2004;48:3720-8.  Back to cited text no. 4
5.Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. CLSI M100-S20U. Wayne, PA: Clinical and Laboratory Standards Institute; 2010. Update 2010 Jun.  Back to cited text no. 5
6.Yan JJ, Wu JJ, Tsai SH, Chuang CL. Comparison of the double-disk, combined disk, and Etest methods for detecting metallo-beta-lactamases in gram-negative bacilli. Diagn Microbiol Infect Dis 2004;49:5-11.  Back to cited text no. 6
7.Pérez-Pérez FJ, Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002;40:2153-62.  Back to cited text no. 7
8.Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 2000;66:4555-8.  Back to cited text no. 8
9.Mohamudha PR, Harish BN, Parija SC. Molecular description of plasmid-mediated AmpC β-lactamases among nosocomial isolates of Escherichia coli & Klebsiella pneumoniae from six different hospitals in India. Indian J Med Res 2012;135:114-9.  Back to cited text no. 9
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11.Peter-Getzlaff S, Polsfuss S, Poledica M, Hombach M, Giger J, Böttger EC, et al. Detection of AmpC beta-lactamase in Escherichia coli: Comparison of three phenotypic confirmation assays and genetic analysis. J Clin Microbiol 2011;49:2924-32.  Back to cited text no. 11
12.Hanson ND, Moland ES, Hong SG, Propst K, Novak DJ, Cavalieri SJ. Surveillance of community-based reservoirs reveals the presence of CTX-M, imported AmpC, and OXA-30 beta-lactamases in urine isolates of Klebsiella pneumoniae and Escherichia coli in a U.S. community. Antimicrob Agents Chemother 2008;52:3814-6.  Back to cited text no. 12
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Correspondence Address:
Arindam Chakraborty
PhD Scholar, Department of Medicine, Kasturba Medical College, Manipal University, Mangalore - 575 001, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.134700

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