Occurrence and detection of AmpC ß-lactamases among Gram-negative clinical isolates using a modified three-dimensional test at Guru Tegh Bahadur Hospital, Delhi, India

Vikas Manchanda* and Narendra P. Singh

Department of Microbiology, University College of Medical Sciences and Guru Tegh Bahadur Hospital, Dilshad Garden, 110095 Delhi, India

Received 1 July 2002; returned 2 August 2002; revised 11 November 2002; accepted 20 November 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
AmpC enzymes can be differentiated from other extended-spectrum ß-lactamases by their ability to hydrolyse cephamycins as well as other extended-spectrum cephalosporins. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Delhi, India. Among the 135 clinical isolates of Gram-negative bacilli tested, 20.7% were found to harbour AmpC enzymes using a modified three-dimensional test. Inhibition of zone distortion in the presence of cloxacillin confirmed AmpC-harbouring isolates. Maximal incidence of AmpC producers was found among Acinetobacter spp. (42.8%) followed by Klebsiella pneumoniae isolates (33.3%). No AmpC-harbouring isolates revealed decreased susceptibility to cefoxitin. Therefore, Gram-negative bacilli showing resistance to any cephalosporin or aztreonam irrespective of cefoxitin susceptibility should be screened for the AmpC enzyme. The modified three-dimensional test is easy to carry out and can be applied as a phenotypic screening method for detection of AmpC-harbouring Gram-negative organisms. This is the first study to determine the occurrence of AmpC ß-lactamases from India.

Keywords: AmpC ß-lactamases, ESBLs, three-dimensional technique, AmpC India


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
AmpC class ß-lactamases are cephalosporinases that are poorly inhibited by clavulanic acid. They can be differentiated from other extended-spectrum ß-lactamases (ESBLs) by their ability to hydrolyse cephamycins as well as other extended-spectrum cephalosporins. Plasmid-encoded AmpC ß-lactamases are produced by numerous pathogens, e.g. Klebsiella pneumoniae, Escherichia coli, Salmonella spp., Proteus mirabilis and Citrobacter freundii.1 Although reported with increasing frequency, the true rate of occurrence of AmpC ß-lactamases in different organisms, including members of Enterobacteriaceae, remains unknown. Coudron et al.2 used the standard disc 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 ß-lactamases. The disc diffusion test was found to be non-specific and there is always a search for newer methods and the aim to make existing methods more user-friendly to detect these enzymes for use in routine diagnostic laboratories. The main aim is to pass on the benefit to the ultimate beneficiary, the patient, as quickly as possible and, obviously, at lowest possible cost. The current National Committee for Clinical Laboratory Standards (NCCLS) guidelines do not describe any method for detection of isolates producing AmpC ß-lactamases.3 The present study was designed to determine the occurrence of AmpC ß-lactamases from India. Moreover, the three-dimensional test was modified, being made user-friendly to be applied as a phenotypic screening method for detection of AmpC-harbouring Gram-negative organisms.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

The study included 135, non-repeat, non-enteric clinical isolates of Gram-negative bacilli collected over a period of 3 months from Guru Tegh Bahadur Hospital in Delhi, India. The isolates were obtained from clinical specimens from the NICU, the ICU, the burns unit, inpatient units and the outpatient department. The organisms included E. coli (49 isolates), K. pneumoniae (27 isolates), Pseudomonas spp. (18 isolates), Citrobacter spp. (15 isolates), Acinetobacter spp. (14 isolates), Enterobacter spp. (nine isolates) and P. mirabilis (three isolates). Isolates were identified with standard biotyping methods.4 Antimicrobial susceptibility testing was carried out with the disc diffusion method using current NCCLS recommendations.3 Commercially available antibiotic discs (Oxoid, Basingstoke, UK) were used. The antimicrobial susceptibility profiles against ciprofloxacin, chloramphenicol, co-trimoxazole, cephalosporins (cefalexin, cefotaxime, ceftazidime, cefpirome and cefoxitin), aztreonam and imipenem were studied.

ß-Lactamases

Irrespective of their antimicrobial susceptibility profile all isolates of E. coli and K. pneumoniae were tested for ESBL production using ceftazidime (30 µg) and cefotaxime (30 µg) discs and clavulanic acid (10 µg/disc) as recommended by the NCCLS.3 Quality control was achieved using K. pneumoniae (ATCC 700603) and E. coli (ATCC 25922). Increase in zone diameter (>=5 mm) for either antimicrobial agent tested in combination with clavulanic acid versus its zone when tested alone was a positive test for ESBL producers.

AmpC enzyme: modified three-dimensional test

AmpC enzyme production was detected by a modified three-dimensional test. Briefly, fresh overnight growth from Mueller–Hinton agar was transferred to a pre-weighed sterile microcentrifuge tube. The tube was weighed again to ascertain the weight of the bacterial mass. The technique was standardized so as to obtain 10–15 mg of bacterial wet weight for each sample. The growth was suspended in peptone water and was pelleted by centrifugation at 3000 rpm for 15 min. Crude enzyme extract was prepared by repeated freeze–thawing. Five rounds of freeze–thawing gave satisfactory results but some live bacteria remained whose growth into the slits sometimes interfered with the results. To ensure complete membrane lysis to minimize the possibility of live organisms and to extract optimal enzyme concentrate, the freeze–thawing was performed seven times. Lawn cultures of E. coli ATCC 25922 were prepared on Mueller–Hinton agar plates and cefoxitin (30 µg) discs were placed on the plate. Linear slits (3 cm) were cut using a sterile surgical blade 3 mm away from the cefoxitin disc. Small circular wells were made on the slits at 5 mm distance, inside the outer edge of the slit, by stabbing with a sterile pasture pipette on the agar surface. The wells could easily be loaded with the enzyme extract in 10 µL increments until the well was filled to the top. Approximately 30–40 µL of extract was loaded in the wells. The plates were kept upright for 5–10 min until the solution dried, and were then incubated at 37°C overnight. The test was repeated with enzyme extract with a 5 µg cloxacillin disc (Oxoid) added to the extract and incubated for 37°C for 30 min. Quality control was achieved using a known AmpC positive isolate of K. pneumoniae, kindly provided by Dr Patricia Bradford, Wyeth Laboratories, New York, NY, USA.

Three different kinds of result were recorded. The isolates showing clear distortion of zone of inhibition of cefoxitin were taken as AmpC producers. The isolates with no distortion were taken as AmpC non-producers and isolates showing minimal distortion were taken as indeterminate strains (Figure 1). Inhibition of zone distortion when cloxacillin discs in enzyme extract were used confirmed AmpC producers.



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Figure 1. Organisms showing clear distortion in the zone of inhibition: strains A (test strain) and B (control strain) were taken to be AmpC producers, minimal distortion (strain C) was noted as indeterminate and no distortion, strain D (negative control) was taken to indicate non-AmpC producers.

 

    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Disc diffusion susceptibility tests and ESBLs

Multiple drug resistance (resistance to three or more drugs) was observed in most of the isolates (76.2%). Resistance to five or more drugs was observed among 67.4% isolates.

K. pneumoniae had the highest occurrence of ESBL producers (92.5%) followed by E. coli (55.1%). Interestingly, there was concurrent resistance to gentamicin and aztreonam in all the ESBL-producing K. pneumoniae and E. coli isolates, whereas resistance towards either of the antibiotics alone was not an indicator of ESBL production.

AmpC enzyme

Twenty-eight isolates were found to harbour AmpC enzyme (20.7%). Use of cloxacillin discs confirmed the presence of AmpC enzyme in all 28 isolates. Maximal incidence of AmpC producers was found among Acinetobacter spp. (42.8%) followed by K. pneumoniae isolates (33.3%). Among all the AmpC-harbouring isolates, those collected from the ICU had the maximal number of AmpC positives (45%) followed by the burns unit (22%). Interestingly, no AmpC-harbouring isolates revealed decreased susceptibility to cefoxitin. Table 1 shows susceptibility patterns of cefoxitin among different AmpC-positive and -negative isolates. Sixty-one per cent of AmpC producers were found to be resistant to cefoxitin.


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Table 1.  Frequency of cefoxitin resistance among AmpC-positive and -negative isolates
 
Three isolates (two E. coli and one Pseudomonas spp.) were ‘indeterminate’ for AmpC enzyme by the three-dimensional test. All three isolates were sensitive to cefoxitin. The isolates were found to be ESBL non-producers and were sensitive to most of the antimicrobials tested.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Various researchers have tried the three-dimensional test with different modifications to detect the AmpC class of ß-lactamases, but, to date, no satisfactory technique has been established. Thomson et al.5 originally described the test. But the technique had its own limitations, namely requirement of a customized low-speed turntable for application of inocula into a circular slit in agar. Subsequently, the test was modified with the use of a rectangular (‘cylindrical’) slit technique with bacterial suspensions and enzyme extracts, but filling these slits was a fastidious procedure.2,6 The technique had advantages in terms of less expertise and minimal equipment required, but great care had to be taken while incubating the plates to prevent spillover of the suspension. Moreover, filling the slits homogeneously without overflow on to the agar surface remained problematic. To overcome these problems we modified the technique. We created a small circular well on the slit at 5 mm distance, inside the outer edge of the slit, using a sterile pasture pipette. This served two main purposes. First, any spillage while loading an enzyme extract was far from the area where distortion of zone had to be observed. Furthermore, filling the slits through wells was easy and faster than the previous techniques.

All the isolates found harbouring AmpC enzyme were further confirmed by the use of cloxacillin discs that showed decreased distortion or complete inhibition of the enzyme. AmpC ß-lactamases are inhibited by cloxacillin, and preparations with IEF patterns that demonstrated the loss of a nitrocefin band after application of a cloxacillin-moistened strip.7

The three-dimensional test can be extremely sensitive; in a study the test did not reveal false negative results and only one of the 28 AmpC harbouring isolates was false positive.2 This suggests that the technique can be used for routine screening of the AmpC enzyme in a clinical laboratory, if made user- friendly, as done with the modifications described above. But the sensitivity of the test has not been confirmed for organisms other than E. coli and Klebsiella spp. In general, cefoxitin readily detects hyperproduction of AmpC in Enterobacter spp. and in C. freundii. A low level of production yields negative results or marginally positive results.

In the present study, 61% of AmpC producers were found to be resistant to cefoxitin and 39% were susceptible to cefoxitin. Interestingly, all of the cefoxitin-susceptible isolates that harboured an AmpC ß-lactamase had MICs of cefoxitin <2 mg/L using the broth dilution method (data not shown). Bauernfeind et al.8 isolated a clinically significant strain of K. pneumoniae that harboured a novel type of AmpC ß-lactamase and that also demonstrated a low level of activity against cephamycins (cefoxitin MIC 4 mg/L). Recently, researchers have found ampC alleles from the chromosomes of two ß-lactam-sensitive C. freundii strains isolated in the 1920s, before the clinical use of antibiotics.9 Cefoxitin resistance in AmpC non-producers could be due to some other resistance mechanism(s). Lack of permeation of porins as one of the resistance mechanisms has been reported.10 Hernandez-Alles et al.11 have demonstrated that interruption of a porin gene by insertion sequences is a common type of mutation that causes loss of porin expression and increased cefoxitin resistance in K. pneumoniae. AmpC production in cefoxitin-susceptible isolates may have a mechanism similar to that of ESBL-producing organisms that appear susceptible to ceftazidime by the disc diffusion method. These data indicate that although screening methods that use cefoxitin in standardized methods to detect AmpC-harbouring isolates are useful, they are not perfect. The results in the present study showed that screening should include all the clinical isolates showing resistance to any of the cephalosporins and/or aztreonam, irrespective of their cefoxitin susceptibility status.

Emerging evidence suggests that probably all Acinetobacter baumannii isolates produce a chromosomal AmpC enzyme.12 In the present study, 42.8% of Acinetobacter spp. were found to harbour an AmpC enzyme. The reason for this low occurrence could be that all the isolates may have ampC genes, but these might not be expressed in all the isolates of Acinetobacter spp. This means they might have ‘silent genes’ or that there might be low level expression of ampC genes that was not detected by the present method. Since only genes that are expressed cause resistance, a phenotypic test like the three-dimensional test may be more valuable than a genotypic method like PCR for such isolates. Moreover, Indian strains of Acinetobacter spp. might be less likely to express ampC genes than Western strains.

In the present study, ESBL-producing isolates of K. pneumoniae and E. coli were isolated from inpatient units as well as from clinical samples from patients attending outpatient clinics. In contrast, all the AmpC-harbouring organisms were found only in clinical specimens from admitted patients, except one isolate (an E. coli isolate from a urinary specimen) from a patient attending outpatient clinics. This clearly shows that at present AmpC-harbouring isolates are largely restricted to hospitalized patients only.

To conclude, the occurrence of ESBLs in K. pneumoniae was quite high, reaching outbreak levels (92.5%). The occurrence of ESBLs in E. coli was also high (55%). Moreover, ESBL-producing strains have spread into the community, whereas AmpC-harbouring organisms are still limited to the hospital. Overall AmpC occurrence was also relatively high (20.7%) compared with earlier reported studies from other countries. The modified three-dimensional test was an easier and rapid screening method for detection of AmpC enzymes.


    Footnotes
 
* Corresponding author. Tel: +91-11-2747-2222; Fax: +91-11-2229-0495; E-mail: manchandavikas{at}hotmail.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Bauernfeind, A., Chong, Y. & Lee, K. (1998). Plasmid-encoded AmpC beta-lactamases: how far have we gone 10 years after the discovery? Yonsei Medical Journal 39, 520–5.[ISI][Medline]

2 . Coudron, P. E., Moland, E. S. & Thomson, K. S. (2000). Occurrence and detection of AmpC beta-lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a Veterans Medical Center. Journal of Clinical Microbiology 38, 1791–6.[Abstract/Free Full Text]

3 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Susceptibility Testing—Ninth Informational Supplement: M100-S10. NCCLS, Wayne, PA, USA.

4 . Crichton, P. B. (1996). Enterobacteriaceae: Escherichia, Klebsiella, Proteus and other genera. In Mackie & McCartney Practical Medical Microbiology, 14th edn (Collee, J. G., Fraser, A. G., Marmion, B. P. & Simmons, A., Eds), pp. 361–84. Churchill Livingstone, Edinburgh, UK.

5 . Thomson, K. S., Mejglo, Z. A., Pearce, G. N. & Regan, T. J. (1984). 3-Dimensional susceptibility testing of beta-lactam antibiotics. Journal of Antimicrobial Chemotherapy 13, 45–54.[Abstract]

6 . Vercauteren, E. P., Descheemaeker, M. I., Sanders, C. C. & Goosens, H. (1997). Comparison of screening methods for detection of extended-spectrum beta-lactamases and their prevalence among blood isolates of Escherichia coli and Klebsiella spp. in a Belgian teaching hospital. Journal of Clinical Microbiology 35, 2191–7.[Abstract]

7 . Sanders, C. C., Sanders, W. E. & Moland, E. S. (1986). Characterization of beta-lactamases in situ on polyacrylamide gels. Antimicrobial Agents and Chemotherapy 30, 951–2.[ISI][Medline]

8 . Bauernfeind, A., Schneider, I., Jungwirth, R., Sahly, H. & Ullmann, U. (1999). A novel type of AmpC beta-lactamase, ACC-1, produced by a Klebsiella pneumoniae strain causing nosocomial pneumonia. Antimicrobial Agents and Chemotherapy 43, 1924–31.[Abstract/Free Full Text]

9 . Barlow, M. & Hall, B. G. (2002). Origin and evolution of the AmpC beta-lactamases of Citrobacter freundii. Antimicrobial Agents and Chemotherapy 46, 1190–8.[Abstract/Free Full Text]

10 . Pangon, B., Bizet, C., Bure, A., Pichon, F., Philippon, A., Ragnier, B. et al. (1989). In vivo selection of a cephamycin-resistant, porin-deficient mutant of K. pneumoniae producing a TEM-3 beta-lactamase. Journal of Infectious Diseases 159, 1005–6.[ISI][Medline]

11 . Hernandez-Alles, S., Benedi, V. J., Martinez-Martinez, L., Pascual, A., Aguilar, A., Tomas, J. M. et al. (1999). Development of resistance during antimicrobial therapy caused by insertion sequence interruption of porin genes. Antimicrobial Agents and Chemotherapy 43, 937–9.[Abstract/Free Full Text]

12 . Bou, G. & Beltran, J. M. (2000). Cloning, nucleotide sequencing and analysis of the gene encoding an AmpC beta-lactamase in Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 44, 428–32.[Abstract/Free Full Text]