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 Table of Contents  
Year : 2016  |  Volume : 21  |  Issue : 2  |  Page : 107-110

AmpC β-lactamases producing Gram-negative clinical isolates from a tertiary care hospital

Department of Microbiology, Dayanand Medical College and Hospital, Ludhiana, Punjab, India

Date of Web Publication31-Aug-2016

Correspondence Address:
Veenu Gupta
Department of Microbiology, Dayanand Medical College and Hospital, Ludhiana- 141 001, Punjab
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0971-9903.189526

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Introduction: AmpC βlactamases are clinically significant since these confer resistance to cephamycins as well as other extended-spectrum cephalosporins and are not affected by βlactamase inhibitors. Objectives: This prospective study was planned to detect AmpC producers among Gram-negative clinical isolates and to compare drug resistance in AmpC and non-AmpC producers. Materials and Methods: In this 1-year prospective study, all Gram.negative isolates were identified by colony characteristics, Gram.staining, biochemical reactions as per standard procedures. Antimicrobial susceptibility was done by as per Clinical and Laboratory standards Institute guidelines. The AmpC was detected by AmpC disc test. Results: Of 2100 samples received, 581 samples showed growth of Gram-negative isolates. Monomicrobial growth was seen in 79.8% and polymicrobial in 20.1%. There were 711 Gram-negative isolates. Of which Acinetobacter baumannii (32.5%), Klebsiella pneumoniae (29.2%), and Pseudomonas aeruginosa (22.3%) were common. Antimicrobial susceptibility pattern showed increased sensitivity toward carbapenems, polymixin B, and tigecycline. AmpC- β-lactamase production was seen in 13.3% isolates and AmpC producers showed high resistance to various antimicrobial agents as compared to non-AmpC producers. Conclusion: Majority of Gram-negative isolates were multidrug resistant and AmpC production was seen in 13.3% isolates. A. baumannii and K.pneumoniae showed maximal AmpC production. AmpC testing is therefore recommended as a mandatory test in a hospital set up.

Keywords: Amp-c-β-lactamase, gram-negative isolates, antimicrobial susceptibility

How to cite this article:
Kaur S, Gupta V, Chhina D. AmpC β-lactamases producing Gram-negative clinical isolates from a tertiary care hospital. J Mahatma Gandhi Inst Med Sci 2016;21:107-10

How to cite this URL:
Kaur S, Gupta V, Chhina D. AmpC β-lactamases producing Gram-negative clinical isolates from a tertiary care hospital. J Mahatma Gandhi Inst Med Sci [serial online] 2016 [cited 2023 Mar 29];21:107-10. Available from: https://www.jmgims.co.in/text.asp?2016/21/2/107/189526

  Introduction Top

Gram-negative bacteria can cause serious infection in hospitalized patients. Treatment of these infections is often complicated because of the increasing bacterial resistance mediated by varying degrees of β-lactamase enzymes. Hydrolysis of β-lactam antibiotics by β-lactamases is the most common mechanism of resistance for this class of antibacterial agents in clinically important Gram-negative bacteria. Because penicillins, cephalosporins, and carbapenems are included in the preferred treatment regimens for many infectious diseases, the presence and characteristics of these enzymes play a critical role in the selection of appropriate therapy. β-Lactamase production is most frequently suspected in a Gram-negative bacterial isolate that demonstrates resistance to a β-lactam antibiotic.[1]

AmpC β-lactamases are cephalosporinases, which belong to the molecular Class C as classified by Ambler in 1980 and Group I under a classification scheme of Bush and Jacoby. These are clinically significant as they may confer resistance to a wide variety of β-lactam drugs, including α-methoxy-β-lactams, narrow, expanded and broad-spectrum cephalosporins, aztreonam, a monobactam and most significantly β-lactam plus β-lactamase inhibitor combinations (viz., ampicillin-clavulanic acid, piperacillin-tazobactam etc).[2],[3],[4],[5]

Therefore, this prospective study was conducted to determine AmpC production in Gram-negative clinical isolates and to compare drug resistance in AmpC and non-producers.

  Materials and Methods Top

All clinical samples (respiratory, blood, pus, urine, etc) received from the patients admitted in surgical Intensive Care Units during 1 year study period (February 2012 to January 2013) were processed as per standard protocol.[6] Further identification and characterization of only Gram-negative isolates were done.

AmpC production: For screening, the Mueller- Hinton agar plate was inoculated using a swab that has been submerged in a bacterial suspension by lawn culture method. Cefoxitin disc (30 µg) was applied and the plates were incubated at 35-37o C for 24 h. Then, zones of inhibition were measured. The organism was labeled as probable Amp- C producer if zone size was <18 mm and was subjected to confirmatory AmpC disc test [Figure 1].
Figure1: Cefoxitin disc test for screening

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AmpC disc test

A lawn culture of Escherichia coli ATCC 25922 was prepared on a MHA plate, and a cefoxitin disc (30 µg) was placed on the plate. AmpC disc was moistened with 20 µl of sterile saline and inoculated with colonies of the test organism. This disc was then placed beside the cefoxitin disc (almost touching) with the inoculated side facing downward. The MHA plate was incubated at 35°C for 24 h. If there was flattening or indentation of cefoxitin inhibition zone, it was considered as an AmpC producer [Figure 2].[7]
Figure2: AmpC disc test for confirmation

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Antimicrobial susceptibility testing was done as per Clinical and Laboratory Standards Institute guidelines [8] and resistance profile of AmpC and non-AmpC producers was compared and put to statistical analysis.

  Results Top

Out of 2100 samples, the majority were respiratory, followed by blood and pus samples. A total of 581 samples showed growth of Gram-negative isolates. Monomicrobial growth was seen in 79.8% and polymicrobial in 20.1%. A total of 711 Gram-negative isolates were obtained. Most common isolate was Acinetobacter baumannii (32.5%) followed by K. pneumoniae (29.2%) and Pseudomonas aeruginosa (22.3%) [Figure 3] Among the respiratory samples most common isolate was A. baumannii (46.1%) whereas K. pneumoniae (44.5%) and E. coli (40.3%) were predominant in blood and pus samples respectively. Gram-negative isolates showed the highest resistance to ampicillin, third generation cephalosporins, cotrimoxazole, piperacillin and ciprofloxacin. Among aminoglycosides, highest resistance was seen toward gentamicin (60.3%). All isolates were moderately resistant to piperacillin-tazobactam and cefoperazone-sulbactam, however showed the least resistance toward imipenem. None of these isolates were resistant to polymixin B and tigecycline [Figure 4].
Figure3: Distribution of Gram-negative organisms(n=711)

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Figure4: Antimicrobial resistance profile of Gram-negative organisms(711)

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AmpC production was seen in 13.3% of Gram-negative isolates. AmpC production was detected in 23.3% A. baumannii isolates followed by K. pneumoniae (14.4%), E. coli (7.8%) and P. aeruginosa (2.5%). Majority of Amp-C producers were from respiratory (56.8%) followed by blood (22.1%), pus (10.5%) and body fluid isolates (8.4%) Amp-C producers showed high degree of resistance to amikacin, ciprofloxacin, piperacillin, third and fourth generation cephalosporins, piperacillin-tazobactam, cefoperazone-sulbactam and imipenem, as compared to non-AmpC producers and significant statistical difference was obtained [Table 1].
Table 1: Comparison of antibiotic resistance profile of AmpC and non-AmpC producers

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  Discussion Top

The 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 protocols. Although CLSI recommendations exist, they are limited to extended- spectrum beta-lactamase -producing E. coli and Klebsiella species. No recommendations exist for ESBL detection and reporting for other organisms and for detection of AmpC β-lactamases. Failure to detect these organisms has contributed to their uncontrolled spread and the consequent clinical failures.[9],[10]

AmpC production was seen in 13.3% isolates in this study as compared to other studies which reported 14.8%, 17.3% and 22% of the isolates as AmpC producers.[5],[11],[12] In this study, AmpC production was highest in A. baumannii (23.3%), followed by K. pneumoniae (14.4%), similar with the study done at Karnataka, which showed 43.5% of Acinetobacter spp. as AmpC producers,[13] whereas Subha et al. found maximum AmpC β-lactamase production in Klebsiella (24.1%) and E. coli (37%).[14]

As evident from the above studies and result of this study, AmpC production among different organisms varies in different institutions. However, still AmpC harboring isolates in clinical specimens of ICU patients is of concern. AmpC producing bacterial pathogens may cause a major therapeutic failure if not detected and reported on time.[15]

In this study, the comparison of antimicrobial resistance profile of AmpC and non-AmpC producers showed a significant statistical difference. AmpC producers showed a higher degree of resistance toward various antimicrobial agents similar with the findings of various authors.[11],[15],[16],[17]

AmpC producing isolates were sensitive to imipenem, thereby reiterating the continued efficacy of carbapenems as the first-line agents for the treatment of nosocomial infections caused by Gram-negative bacteria producing AmpC β-lactamases. The need of the hour is that every health care institution must develop its own antimicrobial stewardship program which is based on the local epidemiological data and international guidelines, to optimize the antimicrobial use among the hospitalized patients, to improve the patient outcomes, to ensure a cost-effective therapy, and to reduce the adverse consequences of the antimicrobial use.

  Conclusion Top

In our study, A. baumannii, K. pneumoniae, and P. aeruginosa were common isolates. Imipenem was most effective drug against these isolates. AmpC production was observed in 13.3% isolates, and AmpC producers were significantly more drug resistant.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Bush K, Jacoby GA. Updated functional classification of beta-lactamases. Antimicrob Agents Chemother 2010;54:969-76.  Back to cited text no. 1
Ambler RP. The structure of beta-lactamases. Philos Trans R Soc Lond B Biol Sci 1980;289:321-31.  Back to cited text no. 2
Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 1995;39:1211-33.  Back to cited text no. 3
Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-type beta-lactamases. Antimicrob Agents Chemother 2002;46:1-11.  Back to cited text no. 4
Arora S, Bal M. AmpC beta-lactamase producing bacterial isolates from Kolkata hospital. Indian J Med Res 2005;122:224-33.  Back to cited text no. 5
Collee JG, Miles RS, Watt B. Tests for identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th ed. London: Churchill Livingstone; 2007.  Back to cited text no. 6
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. 7
Clinical and Laboratory Standard Institute. Performance Standards for Antimicrobial Susceptibility Testing Twenty Second Informational Supplement CLSI Document M-100-S22. Wayne, PA Clinical and Laboratory Standard Institute; 2012. p. 32.  Back to cited text no. 8
Susic E. Mechanisms of resistance in Enterobacteriaceae towards beta-lactamase antibiotics. Acta Med Croatica 2004;58:307-12.  Back to cited text no. 9
Thomson KS. Controversies about extended-spectrum and AmpC beta-lactamases. Emerg Infect Dis 2001;7:333-6.  Back to cited text no. 10
Bandekar N, Vinodkumar CS, Basavarajappa KG, Prabhakar PJ, Nagaraj P. The beta lactamases mediated resistance amongst the gram negative Bacilli in burn infections. Int J Biol Res 2011;2:766-70.  Back to cited text no. 11
Bhattacharjee A, Anupurba S, Gaur A, Sen MR. Prevalence of inducible AmpC beta-lactamase-producing Pseudomonas aeruginosa in a tertiary care hospital in northern India. Indian J Med Microbiol 2008;26:89-90.  Back to cited text no. 12
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Goel V, Hogade SA, Karadesai SG. Prevalence of extended-spectrum beta–lactamases, AmpC beta-lactamases and mettalo-beta-lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii in an intensive care unit in a tertiary care hospital. J Sci Soc 2013;40:28-31.  Back to cited text no. 13
  Medknow Journal  
Subha A, Devi VR, Ananthan S. AmpC beta-lactamase producing multidrug resistant strains of Klebsiella spp. and Escherichia coli isolated from children under five in Chennai. Indian J Med Res 2003;117:13-8.  Back to cited text no. 14
Singh RK, Pal NK, Banerjee M, Sarkar S, SenGupta M. Surveillance on extended spectrum β-lactamase and AmpC β-lactamase producing gram negative isolates from nosocomial infections. Arch Clin Microbiol 2012;3:1-7.  Back to cited text no. 15
Manchanda V, Singh NP. Occurrence and detection of AmpC beta-lactamases among Gram-negative clinical isolates using a modified three-dimensional test at Guru Tegh Bahadur Hospital, Delhi, India. J Antimicrob Chemother 2003;51:415-8.  Back to cited text no. 16
Datta P, Thakur A, Mishra B, Gupta V. Prevalence of clinical strains resistant to various beta-lactams in a tertiary care hospital in India. Jpn J Infect Dis 2004;57:146-9.  Back to cited text no. 17


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1]

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