The isoelectric focusing patterns of ß-lactamases of Yersinia intermedia isolates from aquatic environments

E. Tzelepia, M. Arvanitidoub, L. S. Tzouvelekisc, A. Mavroidia and A. Tsakrisd,*

a Department of Bacteriology, Hellenic Pasteur Institute, 115 21 Athens; b Departments of Hygiene and d Microbiology, Medical School, Aristotle University of Thessaloniki, 54 006 Thessaloniki; c Department of Microbiology, Medical School, University of Athens, 115 27 Athens, Greece

Sir,

ß-Lactamases among Yersinia spp. have been extensively studied only for Yersinia enterocolitica. Generally, the species produces two distinct chromosomal ß-lactamases, a penicillinase and a cephalosporinase, enzymes A and B, respectively.1 It has been shown that Y. enterocolitica isolates of different biotypes may contain both ß-lactamases, but their expression is related to the susceptibility profiles of ß-lactams.2 However, there are limited data related to ß-lactamase content of the species previously called ‘Y. enterocolitica-like’ and in particular of Yersinia intermedia. In addition, studies concerning the ß-lactamase content of yersinias isolated from aquatic environments have not appeared in the literature.

A total of 30 Y. intermedia isolates recovered from various aquatic environments (bathing, river and drinking water samples) and mussels surviving in seawater in the area of northern Greece were included in this study. All the isolates were identified as previously described.3 MICs of ß-lactam antibiotics were determined at 28°C by an agar incorporation method.4 Drugs (ampicillin, co-amoxiclav, carbenicillin, cefoxitin, cephalothin and ticarcillin) were purchased from commercial sources. The susceptibility status was defined according to the NCCLS recommendations.4 Escherichia coli ATCC 25922 was used as reference strain.

Overnight cultures of the Yersinia strains in trypticase soy broth (Oxoid, Basingstoke, UK) were used to prepare cell suspensions containing c. 1 x 1010 cfu/mL. Crude enzyme extracts were prepared by sonication of each suspension and isoelectric focusing (IEF) of crude enzyme preparations was performed on polyacrylamide gels containing ampholytes covering a pH range 3.5–10 (Pharmacia-LKB, Uppsala, Sweden). ß-Lactamases were visualized by overlaying gels with filter paper moistened with a 0.25 mg/mL solution of nitrocefin (Oxoid) in 0.1 M sodium phosphate buffer (pH 7.0) containing dimethylsulphoxide (1% v/v). A mixture of crude enzyme extracts from E. coli strains producing ß-lactamases with known pIs was used as pI marker.

The TableGo shows the 14 susceptibility profiles of the 30 Y. intermedia isolates according to MIC data of the six ß-lactam antibiotics tested. Isolates usually exhibited resistance to carbenicillin (86.7%) and cephalothin (63.3%), while the majority of them were in the intermediate susceptibility category for ampicillin and ticarcillin (63.3%) as well as for cefoxitin (53.3%) and co-amoxiclav (46.7%). Analysis of ß-lactamase content by IEF revealed that all of the isolates produced two distinct ß-lactamases each, irrespective of their ß-lactam susceptibility profiles (TableGo). One of them was visualized as a faint band in the alkaline region of the gel and exhibited pIs varying from 9 to 9.5. It must be noted that this ß-lactamase band was not always visible and repeat IEF gels, loaded with larger quantities of the crude enzyme preparation, were needed for its detection. The second ß-lactamase was usually present as a double band, the front zone of which focused at pIs varying between 5.5 and 6.1 among examined isolates.


View this table:
[in this window]
[in a new window]
 
Table. Susceptibility profile to ß-lactam antibiotics and ß-lactamase content of Yersinia intermedia isolates
 
Cornelis & Abraham,1 in a number of Y. enterocolitica strains of human and animal origin, have characterized two chromosomally mediated enzymes: a constitutive penicillinase with alkaline pI equal to 8–8.2, enzyme A, and an inducible cephalosporinase with acidic pI focusing at 5.3–5.5, enzyme B. Pham et al.5 also studied ß-lactamases of Y. enterocolitica strains isolated in Australia and found that biotype 1 strains produced both ß-lactamases, in contrast to strains of biotypes 3 and 4 which were found to possess only one type of ß-lactamase. In a more recent study,2 it was clarified that Y. enterocolitica isolates of different biotypes contain both ß-lactamases. Some correlation was detected between susceptibility profiles and expression of these enzymes. For Y. intermedia, however, there are no previous studies describing ß-lactamases. In our previous report,3 double-disc testing showed both penicillinase and inducible cephalosporinase activity in all Y. intermedia isolates. These indications were confirmed by IEF, which revealed the presence of two ß-lactamase bands in all cases, even in those isolates classified as sensitive to carbenicillin or to cephalothin on the basis of their MICs. The fact that few isolates were susceptible to carbenicillin, which is labile to hydrolysis by penicillinase A, and some to cephalothin, which is labile to both penicillinase A and cephalosporinase B enzyme types1 is probably due to low production of the respective enzymes. This explanation is further supported by the observation that relative density of the two main ß-lactamase bands detected on IEF gels was not uniform among isolates, when standard crude enzyme quantities had been loaded. ß-Lactamases with acidic pIs were much more pronounced than those with alkaline pIs, which were extremely faint and not consistently visible in all repeat IEF gels. However, the level of production of these ß-lactamases could be affected in part by their efficiency in hydrolysing nitrocefin. In IEF gels, Pham & Bell,6 have also observed that the band of the penicillinase enzymes in IEF gels remained faint, in contrast to the bands of the cephalosporinases, which became stronger when the crude enzymes had been prepared from cultures induced by imipenem.

Differences in susceptibility and ß-lactamase expression within the same Y. enterocolitica biotype have been detected previously.2 Since all of our isolates possibly produce both A- and B-like ß-lactamases, this finding suggests that strain-to-strain differences in ß-lactam susceptibility profiles of yersinias are not necessarily the result of differences in the ß-lactamase types possessed by the various strains. The majority of our isolates exhibited broad patterns of insensitivity, including insensitivity to both penicillin and cephalosporin antibiotics. This is in agreement with previous studies, where the simultaneous presence of a penicillinase and a cephalosporinase has been related to cross-resistance to various penicillins and older cephalosporins.1,5

The variation in pIs among ß-lactamases of Y. intermedia might reflect the type heterogeneity of the yersinias examined. In comparison with earlier reports describing ß-lactamases of Y. enterocolitica isolates,1,5 a broader range of pIs was detected, although identical pI values do not confirm the presence of the same ß-lactamase type. Such differences are unlikely to be due to variations in the IEF performance between laboratories. They seem rather to represent the variety of ß-lactamases produced by Y. intermedia isolates from diverse sources.

Notes

J Antimicrob Chemother 2000; 46: 513–515

* Corresponding author. Tel: +30-31-99-90-91; Fax: +30-31-99-91-49; E-mail: atsakris{at}med.auth.gr Back

References

1 . Cornelis, G. & Abraham, E. P. (1975). ß-Lactamases from Yersinia enterocolitica. Journal of General Microbiology 87, 273–84.[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 . Tzelepi, E., Arvanitidou, M., Mavroidi, A. & Tsakris, A. (1999). Antibiotic susceptibilities of Yersinia enterocolitica and Y. intermedia isolates from aquatic environments. Journal of Medical Microbiology 48, 157–60.[Abstract]

4 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.

5 . 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]

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]