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 3.510 (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 Table 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 (Table
). 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.
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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: 513515
* Corresponding author. Tel: +30-31-99-90-91; Fax: +30-31-99-91-49; E-mail: atsakris{at}med.auth.gr
References
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