Detection and assay of ß-lactamases in clinical and non-clinical strains of Yersinia enterocolitica biovar 1A

Sachin Sharma, Priya Ramnani and J. S. Virdi*

Microbial Pathogenicity Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi—110 021, India

Received 3 March 2004; returned 30 March 2004; revised 12 June 2004; accepted 14 June 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: To detect ß-lactamases (A & B) and extended-spectrum ß-lactamases (ESBLs) in clinical and non-clinical isolates of Yersinia enterocolitica biovar 1A, and to determine their activity in the presence of specific lactamase inhibitors.

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 (2–8 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Yersinia enterocolitica, an important food-borne enteropathogen, is known to cause gastroenteritis, terminal ileitis and mesenteric lymphadenitis. The organism is extremely heterogeneous and has been classified into six biovars, namely 1A, 1B, 2, 3, 4 and 5. These biovars vary in their geographical distribution, the ecological niches from which they are isolated and their pathogenic potential.1 Y. enterocolitica is resistant to ß-lactam antibiotics and is known to produce, in general, two ß-lactamases, namely ß-lactamase A (Bla-A) and ß-lactamase B (Bla-B). Bla-A, a broad spectrum constitutive class A enzyme, is inhibited by clavulanic acid, whereas Bla-B is an inducible class C cephalosporinase and is inhibited by aztreonam and cloxacillin.24 The presence and expression of Bla-A and Bla-B have been reported to vary among biovars of Y. enterocolitica isolated from different regions of the world. Distributed globally, biovar 4 strains of European, Asian, Brazilian and South African origin contain both Bla-A and Bla-B, whereas similar isolates from Australia and New Zealand contain only Bla-A.5 The biovar 2 strains isolated from the USA and Japan have been reported to possess genes for both Bla-A and Bla-B.2 Biovar 1A strains, which are also distributed globally, have been reported to produce Bla-A and ‘Bla-B-like’ enzymes.6 Recently, Stock et al.7 also suggested the existence of ‘Bla-A-like’ enzymes in European biovar 1A strains. Much of the data on biovar 1A strains, however, pertain to European or Australian isolates. Recent studies reported isolation of Y. enterocolitica from diarrhoeic human stools, pigs8 and aquatic sources9 in India. All these strains belonged to biovar 1A. Although the antibiotic susceptibility profiles of these isolates were reported10 nothing is known about the ß-lactamases of these isolates. In view of the paucity of information about ß-lactamases of biovar 1A strains of Y. enterocolitica from continents other than Europe and Australia, we studied ß-lactamases and extended-spectrum ß-lactamases (ESBLs) of such strains isolated from India.


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

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

Mueller–Hinton agar (MHA), Mueller–Hinton 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 plate—with the adjacent edges 22 mm apart—and 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 M–1 cm–1 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Detection of Bla-A and Bla-B

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.


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Table 1. Detection of ß-lactamases (Bla-A, Bla-B) and ESBLs in Y. enterocolitica biovar 1A

 
The appearance of a small zone of inhibition with a 2–8 mm annular radius (10–22 mm diameter) around the ticarcillin 75 µg disc in all clinical and non-clinical strains (except two of aquatic origin) indicated the presence of the Bla-A enzyme. However, synergy, i.e. the appearance of an additional zone of inhibition around the ticarcillin disc towards the co-amoxiclav 3 µg disc was observed in only 34% (63/183) of the isolates of non-clinical origin. No synergy was observed in any of the clinical isolates. Enzyme B was detected in all the 219 strains of Y. enterocolitica biotype 1A by the unequivocal appearance of characteristic flattening of the zone of inhibition around the cefotaxime 5 µg disc adjacent to the imipenem disc, which served as the inducer for enzyme B.

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 32–64 mg/L and MIC90 64–128 mg/L) to the three cephalosporins tested, namely cefixime, cefpodoxime and cefotaxime.


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Table 2. Susceptibility of Y. enterocolitica biovar 1A to selected antibiotics

 
Detection of ESBLs

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.


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Table 3. ß-Lactamase activity and inhibition studies in Y. enterocolitica biovar 1A

 
ß-Lactamase inhibition studies

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The detection of two ß-lactamases, i.e. Bla-A and Bla-B in biovar 1A strains of Y. enterocolitica isolated from clinical and non-clinical sources, is in consonance with the detection of these enzymes in biovar 1A strains isolated from other parts of the world.3 The enzymes were detected both by MIC determinations and the double disc diffusion assays. Four to eight-fold lower MICs of co-amoxiclav, as compared with that of amoxicillin, indicated the presence of Bla-A. Pham and colleagues11 reported the detection of Bla-A based on both the small radius (2–8 mm) around the ticarcillin disc and the appearance of synergy between the ticarcillin and co-amoxiclav discs. In the present study, none of the clinical isolates showed synergy, whereas among the non-clinical isolates synergy was observed in 34% of the isolates. This variability reflects the differential inhibition of enzyme A by clavulanate, indicating heterogeneity in Bla-A. Pham et al.3 reported that Bla-A was detected consistently by the double disc diffusion method. The lack of synergy between ticarcillin 75 µg and co-amoxiclav 3 µg in all clinical and some non-clinical strains in the present study may be due to inadequate diffusion of clavulanate into the cells because it is known that Bla-A is partitioned between extracellular medium and periplasmic space. Thus, inhibition of Bla-A by clavulanate and the consequent appearance of synergy seem to be influenced, among others, by the nature of the cell wall of the isolates. Therefore, lack of synergy in our isolates may be due to the difference in serotypes used in our study as compared with those used by other investigators who reported synergy consistently. Pham et al.3 also reported that detection of inducible enzyme Bla-B by double disc diffusion was inconsistent. However, in the present study, it was found that detection of Bla-B in Y. enterocolitica biovar 1A by double disc diffusion was consistent, as all the 219 isolates showed unequivocal characteristic flattening of the zone of inhibition around the cefotaxime 5 µg disc. This again points towards heterogeneity in the expression of Bla-B in isolates from different parts of the world. Furthermore, low susceptibility of Indian strains to cephalosporins, namely cefixime, cefpodoxime and cefotaxime—as compared with those reported from other parts of the world—corroborate heterogeneity of Bla-B. Pham et al.3 found that induction of Bla-B was variable between different biovars of Y. enterocolitica. Our results suggest that induction of Bla-B was variable within a biovar. This is amply borne out by the observation that Bla-B induction was ~4.4-fold higher in clinical than in non-clinical isolates, even though all belonged to biovar 1A. The observation that Bla-B was induced more strongly in clinical than in non-clinical isolates may be due to the regular exposure of clinical isolates to ß-lactam antibiotics in vivo. In the present investigation, inhibitor studies also revealed heterogeneity in Bla-A and Bla-B of Y. enterocolitica biovar 1A, as indicated by more pronounced inhibition of these enzymes in clinical strains. The difference was statistically significant (P < 0.001) for inhibition by aztreonam and cloxacillin.

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work is supported by a financial grant from the Defense Research & Development Organization (DRDO) to J. S. V. and a Senior Research Fellowship to S. S. by the Indian Council of Medical Research.


    Footnotes
 
* Corresponding author. Tel: +91-11-26879950; Fax: +91-11-26885270; Email: virdi_dusc{at}rediffmail.com


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Bottone, E. J. (1999). Yersinia enterocolitica: overview and epidemiologic correlates. Microbes and Infection 1, 323–33.[CrossRef][ISI][Medline]

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, 1023–7.[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, 951–7.[Abstract/Free Full Text]

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, 19–24.[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, 9–13.[Abstract]

6 . Pham, J. N. & Bell, S. M. (1993). The prevalence of inducible ß-lactamase in clinical isolates of Yersinia enterocolitica. Pathology 25, 385–7.[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, 403–8.[Abstract/Free Full Text]

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, 1353–5.[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, 510–3.[ISI]

10 . Singh, I. & Virdi, J. S. (2003). In vitro antibiotic susceptibilities of Yersinia enterocolitica biotype 1A. World Journal of Microbiology and Biotechnology 20, 329–31.

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, 268–70.[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, 1004–5.[ISI][Medline]

13 . Working Party of the British Society for Antimicrobial Chemotherapy (1991). A guide to antibiotic sensitivity testing. Journal of Antimicrobial Chemotherapy 27, 1–50.[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, 2191–7.[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, 6035–40.[Abstract]





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