Microbial Pathogenicity Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi110 021, India
Received 3 March 2004; returned 30 March 2004; revised 12 June 2004; accepted 14 June 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: The presence of ß-lactamases and ESBLs was detected by disc diffusion in 219 (36 clinical, 183 non-clinical) isolates. ß-Lactamase activity was assayed spectrophotometrically in all 36 clinical and 10 representative non-clinical isolates using nitrocefin as the substrate. Inhibition of ß-lactamases was studied by clavulanic acid, aztreonam and cloxacillin.
Results: Of the 219 isolates, all except two non-clinical isolates indicated the presence of ß-lactamase A (Bla-A) based on the smaller (28 mm) radius of the inhibition zone around the ticarcillin disc. Synergy between ticarcillin and co-amoxiclav discs was, however, observed in only 34% of isolates of non-clinical origin. ß-Lactamase B (Bla-B) was found to be consistently positive among all the clinical and non-clinical isolates, as indicated by its characteristic appearance of flattening of the zone of inhibition around the cefotaxime disc adjacent to an imipenem disc. Bla-B was induced more strongly in clinical than in non-clinical isolates. Inhibition of enzyme A by clavulanic acid, aztreonam and cloxacillin was found to be similar, whereas enzyme B was inhibited more strongly by aztreonam and cloxacillin. None of the isolates showed the unequivocal presence of ESBL.
Conclusion: This is the first report on ß-lactamases of Yersinia enterocolitica biovar 1A from Asia. Y. enterocolitica biovar 1A expressed both Bla-A and Bla-B. Heterogeneity was, however, discerned in the expression of Bla-A and by induction of Bla-B among clinical and non-clinical isolates of Y. enterocolitica biovar 1A.
Keywords: constitutive Bla-A , inducible Bla-B , nitrocefin , synergy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two hundred and nineteen strains of Y. enterocolitica isolated from different sources, namely clinical (36), pig throat (149), pork (4) and aquatic (30) were examined. The details of these strains were reported earlier.8,9 All strains used in this study have been deposited with the Yersinia National Reference Laboratory and the WHO collaborating centre at the Pasteur Institute (Paris), France.
Antibiotics and chemicals
MuellerHinton agar (MHA), MuellerHinton broth (MHB), tryptone glucose yeast extract (TGYE) agar, tryptone soya broth (TSB) and antibiotic discs, namely ticarcillin 75 µg, imipenem 10 µg, co-amoxiclav 30 µg containing 20 µg amoxicillin and 10 µg clavulanic acid, aztreonam 30 µg, ceftazidime 30 µg and ceftriaxone 30 µg were obtained from Himedia, Mumbai (India). Co-amoxiclav 3 µg containing 2 µg amoxicillin and 1 µg clavulanic acid, cefotaxime 5 µg discs and nitrocefin (SR112C) were purchased from Oxoid (Basingstoke, UK). Amoxicillin, cefotaxime, cefixime and cefpodoxime were purchased from Ranbaxy (India), co-amoxiclav (powder) was from GlaxoSmithKline (India), cloxacillin was from Biochem (India), and imipenem and aztreonam were from Merck, Sharp and Dohme (Sydney, Australia) and Bristol Myers Squibb (USA), respectively. Clavulanic acid was a kind gift from Pfizer (Mumbai, India).
Detection of Bla-A and Bla-B by double disc diffusion test
Bla-A11 and Bla-B12 were detected by double disc diffusion tests. Briefly, a loopful of colony was inoculated from TGYE agar plates into saline to obtain A600=0.1 (107 cfu/mL). 2.5 mL of the suspension was inoculated on MHA plates by flooding, excess was removed and the plates were allowed to dry. For detection of Bla-A, discs containing ticarcillin 75 µg and co-amoxiclav 3 µg were placed on the platewith the adjacent edges 22 mm apartand incubated overnight at 28°C. The radii and the diameters of zone of inhibition around ticarcillin 75 µg were recorded. The plates were also observed for the presence of synergy between these discs, which was indicated by augmentation of the zone of inhibition around the ticarcillin 75 µg disc towards the co-amoxiclav 3 µg disc. For detection of Bla-B, discs containing cefotaxime 5 µg and imipenem 10 µg were placed and incubated similarly. The presence of enzyme B was indicated by the characteristic flattening of the zone of inhibition around the cefotaxime disc adjacent to the imipenem disc.
Determination of minimum inhibitory concentration (MIC)
The MICs of five selected antibiotics, namely amoxicillin, co-amoxiclav, cefixime, cefpodoxime and cefotaxime were determined for 46 isolates of Y. enterocolitica by the microbroth dilution technique in MHB, using the methodology described by the working party of the British Society for Antimicrobial Chemotherapy.13
Detection of ESBLs by three-dimensional (3-D) test
Detection of ESBLs was carried out on MHA.14 Test strains were pre-incubated in TSB at 28°C to achieve an optical density equivalent to 0.5 McFarland standard. The MHA plates were inoculated by swabbing with this bacterial suspension. A ß-lactam antibiotic disc (ceftazidime 30, ceftriaxone 30, cefotaxime 30) or aztreonam 30 was placed in the centre of the agar plate and a cylindrical plug of agar (diameter 4 mm) was punched 2 mm away from the edge of the antibiotic disc. This cup, 4 mm in diameter, was filled with 30 µL of the 3-D inoculum (5.0 McFarland standard) of the test strain grown in TSB at 28°C. A disc of co-amoxiclav 30 µg containing 20 µg amoxicillin and 10 µg clavulanic acid was placed on the agar surface opposite the cup and 30 mm (centre to centre) away from the ß-lactam disc. After overnight incubation at 28°C, plates were examined for unequivocal extension of the zone of inhibition around the ß-lactam disc towards the clavulanic acid disc, and distortion of the inhibition zone on the side of the cup. The distortion was such that growth of the test organism appeared within the zone of inhibition, behind the cup and fully reaching the cup.14
ß-Lactamase induction
Forty-six strains of Y. enterocolitica biovar 1A were tested by slight modification of the induction assay reported earlier.2 A bacterial suspension of the test strain was prepared by inoculating loopfuls of the colonies in saline to obtain OD640 =0.5. Two mL of this suspension was added to each of the two Erlenmeyer flasks that contained 18 mL of TSB. The flasks were incubated at 28°C at 150 rpm until an OD640=0.5 ± 0.02 was achieved. The expression of Bla-B was induced by adding imipenem at a final concentration of 0.5 mg/L in one of the flasks, whereas no inducer was added to the other flask used for assay of constitutive enzyme Bla-A. Incubation of both flasks was continued for another 4 h at 28°C at 150 rpm. At the end of this period, 10 mL of the cell suspension was harvested separately from each flask by centrifugation (Sigma Laborzentrifugen, GmbH, Germany) at 18 000 g for 25 min. The pellet was washed three times with chilled phosphate buffer (0.05 M, pH 7.0). It was finally resuspended in 3 mL of buffer and subjected to sonication (Ultrasonics, VCX 750, USA) on ice with three pulses of 30 s each. The clear cell lysate prepared by centrifugation at 24 000 g for 30 min was used to assay the ß-lactamase activity.
ß-Lactamase assay
ß-Lactamase activity was assessed spectrophotometrically by hydrolysis of nitrocefin.15 The assay mixture contained 83 µg of nitrocefin, 167 µg of BSA, 10% glycerol and 0.33 mL (0.6 µg/mL of protein) of cell lysate that contained ß-lactamase in a final volume of 1.5 mL of 50 mM phosphate buffer. ß-Lactamase activity was monitored by measuring the decrease in absorbance at 390 nm for 10 min at 28°C. The enzyme activity was expressed as µmol of nitrocefin hydrolysed/min/mg of protein and the calculation was based upon the molar extinction coefficient of 15 000 M1 cm1 for nitrocefin.15
ß-Lactamase inhibition studies
Clavulanic acid, aztreonam and cloxacillin (2 mg/L stock solution for each) were used as inhibitors. For inhibition studies, 200 µg of each inhibitor was incorporated into the assay mixture separately and the ß-lactamase activity measured as described above.
Statistical analysis
Statistical analysis was done by the unpaired t-test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of the detection of ß-lactamases Bla-A and Bla-B in clinical and non-clinical (pig throat, pork and aquatic) isolates of Y. enterocolitica biovar 1A are summarized in Table 1.
|
Minimum inhibitory concentration (MIC)
Table 2 shows that the MIC50 of amoxicillin was 512 mg/L for clinical and 1024 mg/L for non-clinical isolates, whereas the MIC90 was 1024 mg/L for both clinical and non-clinical isolates. The MIC50 of co-amoxiclav was 128 mg/L for both clinical and non-clinical isolates, whereas MIC90 was 128 mg/L and 256 mg/L for clinical and non-clinical isolates, respectively. All isolates were also resistant (MIC50 3264 mg/L and MIC90 64128 mg/L) to the three cephalosporins tested, namely cefixime, cefpodoxime and cefotaxime.
|
All clinical and non-clinical isolates were found to be negative for ESBLs by the three-dimensional test (Table 1). No clear-cut zone of extension was observed around any of the four antibiotic discs tested. The characteristic heart-shaped distortion of the zone of inhibition around these discs could be detected in none of the strains, although equivocal distortions were seen in some isolates.
ß-Lactamase induction and activity
The results of induction and inhibition of ß-lactamase activity are shown in Table 3. The mean ß-lactamase activity of uninduced cultures, which indicated the enzyme Bla-A, was 0.75 ± 0.14 and 0.58 ± 0.04 µmol/min/mg of protein for clinical and non-clinical isolates, respectively. This indicated that although Bla-A activity was lower in non-clinical than clinical isolates, it was not statistically significant (P > 0.05). The ß-lactamase activity of the induced cultures, which indicated the activity of enzyme Bla-B, was 3.3 ± 0.38 µmol/min/mg of protein for clinical isolates. This was significantly (P < 0.001) higher than that of the non-clinical (0.75 ± 0.07 µmol/min/mg of protein) isolates. The induction ratio indicated that Bla-B was induced significantly (P < 0.05) more strongly in clinical isolates than in non-clinical isolates.
|
Inhibition studies revealed that enzyme Bla-A (uninduced cultures) of both clinical and non-clinical strains was inhibited uniformly by clavulanic acid, aztreonam and cloxacillin, although inhibition was more pronounced in the clinical isolates. The Bla-B (induced cultures) was, however, inhibited more strongly by aztreonam and cloxacillin than clavulanic acid. Also, for Bla-B, inhibition was more pronounced in clinical than in non-clinical isolates.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In conclusion, biovar 1A strains of Y. enterocolitica isolated from clinical and non-clinical sources were found to produce both Bla-A and Bla-B. None of the strains, however, produced ESBL. The Bla-B was induced more strongly in clinical than in non-clinical strains. Bla-A was inhibited equally by clavulanic acid, aztreonam and cloxacillin. Bla-B, however, was inhibited more strongly by aztreonam and cloxacillin than clavulanic acid.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Stock, I., Heisig, P. & Wiedemann, B. (1999). Expression of ß-lactamases in Yersinia enterocolitica strains of biovars 2, 4 and 5. Journal of Medical Microbiology 48, 10237.[Abstract]
3
.
Pham, J. N., Bell, S. M., Martin, L. et al. (2000). The ß-lactamases and ß-lactam antibiotic susceptibility of Yersinia enterocolitica. Journal of Antimicrobial Chemotherapy 46, 9517.
4 . Pham, J. N., Bell, S. M. & Lanzarone, J. Y. M. (1991). A study of the ß-lactamases of 100 clinical isolates of Yersinia enterocolitica. Journal of Antimicrobial Chemotherapy 28, 1924.[Abstract]
5 . Pham, J. N., Bell, S. M., Hardy, M. J. et al. (1995). Susceptibility to ß-lactam agents of Yersinia enterocolitica biotype 4, serotype O3 isolated in various parts of the world. Journal of Medical Microbiology 43, 913.[Abstract]
6 . Pham, J. N. & Bell, S. M. (1993). The prevalence of inducible ß-lactamase in clinical isolates of Yersinia enterocolitica. Pathology 25, 3857.[ISI][Medline]
7
.
Stock, I., Heisig, P. & Wiedemann, B. (2000). ß-Lactamase expression in Yersinia enterocolitica biovars 1A, 1B and 3. Journal of Medical Microbiology 49, 4038.
8 . Singh, I., Bhatnagar, S. & Virdi, J. S. (2003). Isolation and characterization of Yersinia enterocolitica from diarrheic human subjects and other sources. Current Science 84, 13535.[ISI]
9 . Sinha, I., Choudhary, I. & Virdi, J. S. (2000). Isolation of Yersinia enterocolitica and Yersinia intermedia from wastewater and their biochemical and serological characteristics. Current Science 79, 5103.[ISI]
10 . Singh, I. & Virdi, J. S. (2003). In vitro antibiotic susceptibilities of Yersinia enterocolitica biotype 1A. World Journal of Microbiology and Biotechnology 20, 32931.
11 . Pham, J. N., Bell, S. M., Martin, L. et al. (1999). The detection of enzyme A of Yersinia enterocolitica by disc diffusion method. Pathology 31, 26870.[CrossRef][ISI][Medline]
12 . Pham, J. N. & Bell, S. M. (1993). The detection by a disc diffusion technique of inducible ß-lactamase in Yersinia enterocolitica. Journal of Antimicrobial Chemotherapy 31, 10045.[ISI][Medline]
13 . Working Party of the British Society for Antimicrobial Chemotherapy (1991). A guide to antibiotic sensitivity testing. Journal of Antimicrobial Chemotherapy 27, 150.[ISI][Medline]
14 . Vercauteren, E., Descheemaekar, P., Ieven, M. 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 Belgian teaching hospitals. Journal of Clinical Microbiology 35, 21917.[Abstract]
15 . Perez-Llarena, F., Martin, J. F., Galleni, M. et al. (1997). The bla gene of the cephamycin cluster of Streptomyces clavuligerus encodes a class A ß-lactamase of low enzymatic activity. Journal of Bacteriology 179, 603540.[Abstract]
|