In Finland since 1988 there has been a rapid and substantial increase in resistance to erythromycin in group A streptococci. The extent of this resistance is particularly serious since there are only a few alternative antibiotics available for peroral treatment of group A streptococcal infections.

The nucleotide sequence of PSE-2 beta-lactamase, an enzyme that readily hydrolyzes both carbenicillin and oxacillin, has been determined. The deduced sequence of 266 amino acids contained 93 residues identical to those of OXA-2 beta-lactamase and the Ser-Thr-Phe-Lys tetrad also found in the active site of TEM-1 beta-lactamase.

P-Lactamase identffication by colony hybridization with 32P-labeled DNA probes for TEM-1, SHV-1, OXA-1, OXA-2, PSE-1, PSE-2, and PSE-4 was compared with isoelectric focusing in 122 clinical isolates making a variety of enzyme types. All strains producing a probe-type enzyme gave a positive hybridization reaction. Cross-hybridization was observed between TEM-1 and TEM-2 or TLE-1, between SHV-1 and SHV-2, between OXA-1 and OXA-4, between OXA-2 and OXA-3 (weak), between PSE-2 and OXA-6 or OXA-5 (weak), and among PSE-1, PSE-4, and CARB-3. With allowance for such cross-hybridization, only six strains gave false-positive reactions, and the procedure was 99% specific.Biochemical studies have established that plasmids in gram-negative bacteria determine a variety of P-lactamase types. The enzymes can be differentiated by substrate profile, reactions with inhibitors, molecular weight, and particularly isoelectric point (pl) into at least 25 kinds (16,19,32). Some, such as TEM-1 and SHV-1, have broad-spectrum but relatively unspecialized activity. As their name implies, the OXA set hydrolyzes oxacillin and related P-lactams with particular efficiency, whereas CARB enzymes, also given PSE designations because they were first found in Pseudomonas aeruginosa, are notably effective in carbenicillin hydrolysis. Distinguishing between the enzymes is of value for epidemiological studies where the presence of a less common P-lactamase type may allow a particular strain or plasmid to be traced (20). The distribution of P-lactamase types in different organisms may also help elucidate where these enzymes originated and how they have spread in response to the use and elaboration of P-lactam antibiotics.Studies relying on isoelectric focusing have demonstrated TEM-1 as the predominant P-lactamase in ampicillin-resistant Escherichia coli and Salmonella spp., whereas SHV-1 is preponderant in Klebsiella spp. (29). In P. aeruginosa carbenicillin or ticarcillin resistance due to ,-lactamase production is the result mainly of PSE-1 or PSE-4 (24, 33).If P-lactamase genes are as diverse as the enzymes they encode, it should be possible to develop DNA probes for P-lactamase identification that would allow more rapid studies with large numbers of strains. DNA fragments suitable as probes for TEM-1 (3, 12, 22), OXA-1 (12, 23), OXA-2 (2), and PSE-1 (12) have been described and have been studied for specificity by utilizing purified plasmid DNA. The purpose of this investigation was to apply such probes to clinical isolates to evaluate their sensitivity and specificity. MATERIALS AND METHODSBacterial strains. strains were demonstrated to grow on plates containing 25 ,ug of ampicillin per ml and to produce ,-lactamase as determined by the nitrocefin test (21). Because no probetype P-lactamases were detected among P. aeruginosa isolates from this source, 20 P. aeruginosa clinical isolates with known plasmid-mediated P-lactamases collected by A. Philippon in France before 1981 were also included in hybridization studies.Analytical isoelectric focusin...

We report fingernail onychomycosis caused by Trichophyton equinum in a farmer who breeds racehorses. In addition to the thumbnail, T. equinum had infected one of the racehorses. Oral terbinafine cured the infection in the farmer.

A modified isoelectric focusing method for rapid semiquantitative identification of plasmid-mediated 1-lactamases by use of the Pharmacia PhastSystem (Uppsala, Sweden) is described. Sonicatfon of bacterial colonies collected directly from growth plates decreased the time required for the procedure. With sonic extracts of known (3-lactamase-producing strains used as controls, the assay could be completed in less than 2 h.Isoelectric focusing (IEF) is one of the basic methods for identification and differentiation of plasmid-mediated ,-lactamases (2, 3). We recently described a method for largescale screening for plasmid-encoded ,-lactamase genes that is based on DNA hybridization (1). For studies of small numbers of ,-lactamase-producing strains, however, IEF has proven to be more convenient. In 1975, Matthew et al. used this system for detection and identification of ,-lactamases (2). By using a chromogenic substrate, nitrocefin (2), this system permits visual comparison of patterns of P-lactamase bands produced by enzymes from different organisms. With this method, bands and their satellites can be easily recognized and compared.Quantitative P-lactamase assays are rapid, routine methods in clinical bacteriological laboratories (6). Although classic ,-lactamase IEF assays require 20 to 40 h to complete (2), IEF assays can now be performed in 1 to 1.5 h with certain equipment. In this note, I describe a rapid, semiquantitative IEF method for detection of 1-lactamase that uses PhastSystem (Pharmacia, Uppsala, Sweden) and commercial PhastGels (Pharmacia). This procedure requires minimal amounts of samples and developing reagent.The legend to Fig. 1 lists the 19 control strains (i.e., strains producing known 13-lactamases) that were used (1). In addition, 47 clinical ampicillin-resistant, 3-lactamase-producing isolates (36 Escherichia coli strains, 5 Pseudomonas aeruginosa strains, 5 Klebsiella pneumoniae strains, and 1 Serratia marcescens strain) were included. The presence of P-lactamase-encoding genes was also confirmed by DNA hybridization in all of the clinical isolates studied (1).Crude extracts of bacterial strains were prepared by sonication of cultures grown overnight in brain heart infusion broth and frozen at -20°C until used (2). To develop a more rapid method for preparing sonic extracts, strains were grown on Muller-Hinton agar containing ampicillin (25 jig/ ml) for 18 h at 37°C; cells were scraped from the surface of each plate, suspended in 0.5-ml volumes of 50 mM sodium phosphate buffer (pH 7.0), sonicated, and centrifuged as described by Reid et al. (5).PhastGel IEF media are homogeneous polyacrylamide gels (43 by 50 mm) containing Pharmalyte carrier ampholytes (Pharmacia). Pharmalyte generates stable, linear pH gradients with a smooth conductivity profile across the entire pH range, which means that high field strengths of 500 V/cm and above can be used. Three PhastGel IEF media, identified according to pH gradients, are available: PhastGel IEF 3-9, PhastGel 4-6.5, and PhastGel 5-8. The gels to be...