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
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Abstract |
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Keywords: AmpC ß-lactamases, ESBLs, three-dimensional technique, AmpC India
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Introduction |
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Materials and methods |
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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 MuellerHinton 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 1015 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 freezethawing. Five rounds of freezethawing 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 freezethawing was performed seven times. Lawn cultures of E. coli ATCC 25922 were prepared on MuellerHinton 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 3040 µL of extract was loaded in the wells. The plates were kept upright for 510 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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Results |
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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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Discussion |
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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.
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Footnotes |
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References |
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2
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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, 192431.
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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, 9379.
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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, 42832.