Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh202002, Uttar Pradesh, India
Received 21 March 2004; returned 10 May 2004; revised 20 June 2004; accepted 1 July 2004
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Abstract |
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Methods: ESBL and AmpC producing and non-producing isolates of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, as identified by the conventional three-dimensional extract test, were used to evaluate the modified procedures. Whole bacterial cells and freezethaw preparations, as ß-lactamase sources, were strategically applied to culture plates near ceftazidime and cefoxitin discs on a lawn inoculum of E. coli ATCC 25922. Technical variations of the test included placing the ß-lactamase-containing inoculum into slits, wells and trenches, or onto the surface as spots at varying distances from the discs, and adding clavulanate or cloxacillin to the three-dimensional inoculum to confirm the presence of ESBLs and AmpC ß-lactamases, respectively.
Results: All the methods adopted for ESBL and AmpC detection by using the whole bacterial cells gave positive results. However, the best results were given by the spot-inoculation method. In modifications using the enzymic extracts, the enhanced growth of surface organisms was better appreciated in the designed modifications compared with the conventional methods.
Conclusions: The method described here is simple and cost-effective. Furthermore, up to 16 isolates may be tested on a single culture plate, thus it is a less labour-intensive and more economic technique than other reported phenotypic methods.
Keywords: direct-spot test , well-extract test , modified enzymic extraction , modifications , user-friendly method
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Introduction |
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Double disc-synergy (DDST) and three-dimensional extract tests (TDET) have been used for the detection of ESBLs in members of the family Enterobacteriaceae.2 The current NCCLS documents do not indicate the screening and confirmatory tests that should be used for the detection of AmpC ß-lactamases. However, various workers1,3 have designed the TDET for the detection of AmpC enzymes in Gram-negative bacterial isolates. As the TDET is a labour-intensive procedure, there is always a search for newer methods and the aim to make existing methods more user-friendly to incorporate them in routine diagnostic laboratories.3 Here, we designed a user-friendly and effective protocol to be applied as phenotypic characterization for the detection of ESBLs and AmpC enzymes, and compared our results with the existing phenotypic test, i.e. the conventional three-dimensional extract test.
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Materials and methods |
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Bacterial isolates
A total of 18 AmpC producing isolates (Escherichia coli, 5; Klebsiella pneumoniae, 5; Pseudomonas aeruginosa, 8), 10 AmpC indeterminate isolates (E. coli, 4; K. pneumoniae, 2; P. aeruginosa, 4) and 10 AmpC negative isolates (E. coli, 5; K. pneumoniae, 5), as identified by conventional TDET, were included to detect the AmpC enzyme by the modified procedures. Twenty ESBL producing isolates (E. coli, 10; K. pneumoniae, 10), 10 ESBL indeterminate isolates (E. coli, 5; K. pneumoniae, 5) and 10 ESBL negative isolates (E. coli, 5; K. pneumoniae, 5), inferred by conventional TDET, were also included to detect ESBL production by a modified protocol. Quality control was achieved using a known AmpC positive isolate of K. pneumoniae, obtained from UCMS & GTB Hospital, Delhi, India, originally provided by Dr Patricia A. Bradford, Wyeth Laboratories, Pearl River, NY, USA and a known ESBL producing isolate of K. pneumoniae obtained from RAK Institute of Agricultural Sciences, Aligarh, India. The zone diameters of all the bacterial isolates included in the study were recorded and MICs of cefoxitin and ceftazidime were recorded for isolates with zone diameters less than 18 mm.
ESBL and AmpC enzyme detection by conventional TDET
The isolates were first tested by conventional TDET for the production of ESBL and AmpC enzymes. The methods adopted were of Thomson et al.4 for ESBL detection and Coudron et al.,1 with some modifications as adopted in our previous work,6 for AmpC detection. Briefly, 50 µL of a 0.5 McFarland bacterial suspension prepared from an overnight blood agar plate was inoculated into 12 mL of tryptic soy broth and the culture was grown for 4 h at 35°C. The cells were concentrated by centrifugation, and crude enzyme preparations were made by freezethawing the cell pellets five times. The surface of the MuellerHinton agar plate was inoculated with E. coli strain (ATCC 25922). Then 30 µg cefoxitin (HiMedia, Mumbai, India) and 30 µg ceftazidime (HiMedia) discs were placed on inoculated plates for detection of AmpC enzymes and ESBLs, respectively. With a sterile scalpel blade, a slit beginning 5 mm from the edge of the disc was cut within the agar in an outward radial direction. By using a pipette, 2530 µL of enzyme preparation was dispensed into the slit, beginning near the disc and moving outward. Slit overfill was avoided. The inoculated media were incubated overnight at 35°C. A parallel test was carried out with a 5 µg cloxacillin disc (HiMedia) (for AmpC confirmation) and 10 µg co-amoxiclav disc (HiMedia) (for ESBL confirmation) added to the enzymic extract and the extract incubated at 37°C for 30 min. The whole procedure was then adopted as described above and the parallel sets of plates were incubated overnight at 35°C. Inhibition of zone distortion, when cloxacillin and co-amoxiclav discs in enzyme extract were used, confirmed AmpC and ESBL producers, respectively.
ESBL and AmpC enzyme detection by modified procedures
Cefoxitin (30 µg) and ceftazidime (30 µg) discs were used for AmpC enzyme and ESBL detection, respectively. E. coli ATCC 25922 was used to inoculate the MuellerHinton agar plate. Several modifications were attempted. Broadly, the methods were divided into two groups: (i) methods for AmpC and ESBL detection after deriving the enzymic extracts; (ii) methods using the bacterial isolates directly, rather than using the extracts.
Methods directly using the bacterial isolates
Lawn cultures of E. coli ATCC 25922 were prepared on MuellerHinton agar and the plates were incubated for 15 min at 37°C, for drying. The adopted modifications (Figure 1a) were: (i) with a sterile scalpel blade, a linear cut was made beginning 5 mm from the edge of the discs in an outward radial direction. Eight to 10 distinct colonies were picked with a sterile wire loop and the slit was inoculated at depth, beginning near the disc and moving outwards; (ii) a heavy inoculum was streaked over the agar surface in a linear fashion, beginning 5 mm from the disc and moving outwards; (iii) five to six colonies were spot inoculated at a distance of 78 mm from the edge of the disc; and (iv) five to six colonies were spot inoculated at a distance of 1012 mm. Cefoxitin and ceftazidime discs were placed centrally on the respective plates and overnight incubation at 37°C was carried out. All the results were replicated in at least three separate experiments.
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Approximately, 1015 mg of bacterial wet weight was scraped from the culture plate using a sterile wire loop and suspended in 0.5 mL of peptone water in a sterile microcentrifuge tube and incubated at 37°C for 1 h. Crude enzyme extract was prepared by thrice repeated freezethawing. However, to ensure complete membrane lysis, freezethawing may be carried out five times. Lawn cultures of E. coli ATCC 25922 were prepared as described previously and cefoxitin (30 µg) and ceftazidime (30 µg) discs were placed on respective plates. With a sterile scalpel blade, a slit beginning 5 mm from the edge of the disc was cut in the agar in an outward radial direction, as described in conventional methods, to serve as a control for comparison. Figure 1(b) shows the modifications: (i) a linear trench (3 cm x1 mm) was prepared in the agar; (ii) a circular well made with the broad end of a Pasteur pipette, at a distance of 78 mm from the edge of the cefoxitin/ceftazidime disc; and (iii) a circular well made at a distance of 1012 mm. Fifty microlitres of enzyme preparation was dispensed in trench and wells. However, 30 µL of the enzyme preparation could be loaded in the slit made for the conventional method and slit overfill was avoided. The inoculated media were incubated overnight at 37°C. Enhanced growth of the surface organism at the point where the slit/trench intersected the zone of inhibition or around the wells towards the cefoxitin and ceftazidime discs was interpreted as evidence for the presence of AmpC and ESBLs, respectively. Inhibition of zone distortion when cloxacillin or co-amoxiclav discs in enzyme extracts were used confirmed AmpC and ESBL producers, respectively.
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Results and discussion |
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The bacterial isolates (AmpC or ESBL producers and indeterminate isolates, as inferred by conventional TDET) used in this work either did not have or had very small zones of inhibition (ranging between 8 and 10 mm zone diameters). The MICs (mg/L) of cefoxitin and ceftazidime ranged between 256 and >1024. The bacterial isolates (ESBL producers and ESBL indeterminate) used in this study fulfilled the NCCLS screening criteria5 for ESBL [i.e. MIC of at least one extended-spectrum cephalosporin (ceftazidime was used for screening and confirmation) 2 mg/L] and were positive for the NCCLS ESBL-confirmatory test5 (i.e. MIC of ceftazidime, by broth microdilution method, decreased by
3 doubling dilutions in the presence of clavulanic acid).
AmpC/ESBL detection directly using bacterial isolates
Though all the methods adopted in this study gave positive results, the most discriminatory results were given either by direct inoculation of the organism in the linear cut made in agar or by direct spot inoculation of the test organisms (Figure 1a). Of these, the best results were given by spot inoculation. Therefore, all the bacterial isolates included in this study were screened only by spot inoculation of the test organisms near cefoxitin and ceftazidime discs. All the ESBL and AmpC producing isolates were detected correctly by adopting this method. Nine of 10 indeterminate isolates for AmpC, and eight of 10 indeterminate isolates for ESBL were identified as AmpC and ESBL producers, respectively, and the remaining were identified as indeterminate by this modification. It must be emphasized here that the indeterminate result of conventional TDET looked like a weaker positive result. All AmpC and ESBL-negative isolates gave negative results with the three-dimensional extract test.
AmpC/ESBL detection using enzymic extracts
In the modifications for enzymic extracts, all the 18 AmpC and 20 ESBL producing bacterial isolates were detected correctly by the designed modifications (trenches/wells). Comparing the modifications with the conventional slit test, the enhanced growth of surface organisms was interpreted more easily in the designed modifications (Figure 1b and c). All the isolates, inferred as AmpC and ESBL indeterminate by the conventional method, could be detected as AmpC and ESBL producers by adopting the designed modifications. All the isolates negative for ESBL and AmpC enzymes were detected correctly by the modified procedures.
Among the modifications designed for the test using the enzymic extracts, we have shown that the results with preparation of trenches and wells were comparatively far better than the original slit method as samples inferred to be indeterminate by the slit technique could be detected as producers by the current method. This could be due to the capability of loading large amounts of extract and greater diffusion towards discs, in trenches and wells, than in slits. Another reason for inferring large number of isolates as indeterminate by conventional TDET could be that slight spillage of the extract at the edge of slits, near the discs, could interfere with the proper visualization of the distortion of zone of inhibition. This problem is avoided in our modifications. The preparation of wells was found to be more convenient and easy than trench preparation, as preparation of the trench required extra skill. All the methods described earlier used slit preparation, because, whatever the zone of inhibition for E. coli ATCC 25922 could be, it must intersect the prepared slit. The same is true for trench preparation. To determine the placement of wells, we designed a few more experiments. The zone of inhibition of cefoxitin and ceftazidime discs for the inoculated E. coli ATCC 25922 was noted and the wells were punched within the zone of inhibition (78 mm from the edge of discs), at the zone of inhibition (1012 mm) and outside the margin of the zone of inhibition (Figure 1b). Positive results were obtained with wells punched within, and near the zone of inhibition (Figure 1b and c). The methods for extract preparation described in the previous techniques are lengthy and labour-intensive. Therefore, we tried the modifications that are simple and limit the enzyme extraction time (see the Materials and methods section). In addition, the tests were modified to use direct inoculation of the test bacteria, and excellent results were obtained by inoculating the organism in the depth of the slit in agar and also by spot inoculation (Figure 1a). However, spot inoculation was more convenient. The same experimental design was followed, as for punched wells, to judge the optimal distance for spot inoculations (Figure 1a,). The main advantage of testing the organism by spot inoculation or extracts filled in punched wells is that multiple organisms can be tested on a single plate. We thus propose a more user-friendly protocol for phenotypic detection of ESBLs and AmpC enzymes. (i) Test the organisms for ESBL and AmpC enzyme production by spot inoculating the organisms around ceftazidime and cefoxitin discs, respectively. (ii) Though ß-lactamase detection by spot inoculation was observed to be fairly sensitive in this work, ESBL/AmpC ß-lactamase production could be confirmed by the well test (enhancement of the growth of surface organism near the well filled with extract, and inhibition/reduction in the enhanced growth in the well filled with extract treated with clavulanic acid/cloxacillin). (iii) The organisms found to be negative by the spot test, but suspected of being an ESBL/AmpC producer on the basis of susceptibility or MIC testing should be confirmed by the well extract test.
Diagnostic laboratories demand cheaper and easy to use tests for detection of ESBLs and AmpC enzymes.10 Additionally, there is recent emphasis on medical cost cutting and downsizing at a time when bacterial pathogens are increasing in complexity.10 The current protocol is simple in terms of direct spot inoculation, short enzyme extraction steps, and a simple procedure for the well extract test, without any requirement for sophisticated equipment and extra skills. It should also be noted that detection of ESBL and AmpC enzymes by the spot inoculation test cuts short the reporting time by 1 day as this test could easily be incorporated in the routine diagnostic procedures and the test can be carried out alongside the first-line susceptibility testing. Furthermore, up to 16 bacterial isolates can be screened for ESBL/AmpC production by spot inoculation test or well extract test on a single culture plate. Thus, it is more cost-effective and less labour intensive than other existing phenotypic tests. We suggest that the protocols described here for detection of ESBLs and AmpC enzymes could be incorporated in diagnostic laboratories along with other routine diagnostic procedures.
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Acknowledgements |
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Footnotes |
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References |
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2 . Thomson, K. S. & Sanders, C. C. (1992). Detection of extended-spectrum ß-lactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three-dimensional tests. Antimicrobial Agents and Chemotherapy 36, 187782.[Abstract]
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Manchanda, V. & Singh, N. P. (2003). 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. Journal of Antimicrobial Chemotherapy 51, 4158.
4 . Thomson, K. S., Mejglo, Z. A., Pearce, G. N. et al. (1984). 3-Dimensional susceptibility testing of ß-lactam antibiotics. Journal of Antimicrobial Chemotherapy 13, 4554.[Abstract]
5 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing: Ninth Informational Supplement (M100-S9). NCCLS, Wayne, PA, USA.
6 . Shahid, M., Malik, A. & Sheeba. (2003). Multidrug-resistant Pseudomonas aeruginosa strains harbouring R-plasmids and AmpC ß-lactamases isolated from hospitalised burn patients in a tertiary care hospital of North India. FEMS Microbiology Letters 228, 1816.[CrossRef][ISI][Medline]
7 . Brun-Buisson, C., Legrand, P., Philippon, A. et al. (1987). Transferable enzymatic resistance to third-generation cephalosporins during nosocomial outbreak of multiresistant Klebsiella pneumoniae. Lancet ii, 3026.[CrossRef]
8 . Jarlier, V., Nicolas, M. H., Fournier, G. et al. (1988). Extended broad-spectrum ß-lactamases conferring transferable resistance to newer ß-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Review of Infectious Diseases 10, 86778.[ISI][Medline]
9 . Vercauteren, E. P., Descheemaeker, M. I., Sanders, C. C. et al. (1997). Comparison of screening methods for detection of extended-spectrum ß-lactamases and their prevalence among blood isolates of Escherichia coli and Klebsiella spp. in a Belgian teaching hospital. Journal of Clinical Microbiology 35, 21917.[Abstract]
10 . Thomson, K. S. (2001). Controversies about extended-spectrum and AmpC ß-lactamases. Emerging Infectious Diseases 7, 3336.[ISI][Medline]
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