Characterization of a chromosomal gene encoding type B beta-lactamase in phage group II isolates of Staphylococcus aureus

Abstract

In contrast to most Staphylococcus aureus isolates in which the gene for staphylococcal beta-lactamase (blaZ) is plasmid borne, isolates typeable by group II bacteriophages frequently carry blaZ on the chromosome. Furthermore, the chromosomal gene encodes the type B variant of staphylococcal beta-lactamase for which the nucleotide and deduced amino acid sequences have not yet been reported. To better understand beta-lactamase production among phage group II staphylococci and the nature of the type B beta-lactamase, we determined the type and amount of enzyme produced by 24 phage group II isolates. Of these isolates, 1 did not produce beta-lactamase, 8 produced the type B enzyme, and 15 produced the type C enzyme. In all eight type B beta-lactamase-producing isolates, blaZ was located on the chromosome. This was in contrast to the type C beta-lactamase-producing isolates, in which blaZ was located on a 21-kb plasmid. The nucleotide sequence corresponding to the leader peptide and the N-terminal 85% of the mature exoenzyme form of type B S. aureus was determined. The deduced amino acid sequence revealed 3 residues in the leader peptide and 12 residues in the exoenzyme portion of the beta-lactamase that differ from the prototypic type A beta-lactamase sequence. These include the serine-to-asparagine change at residue 216 found in the kinetically similar type C enzyme, a threonine-to-lysine change at residue 128 close to the SDN loop (residues 130 to 132), and several substitutions not found in any of the other staphylococcal beta-lactamases. In summary, modern isolates of S. aureus typeable by group II phages produce type B or type C staphylococcal beta-lactamase. The type B gene resides on the chromosome and has a sequence that, when compared to the sequences of the other staphylococcal beta-lactamases, corresponds well with its kinetic properties.

FIG. 1

Plot of the ratios of the rate of hydrolysis of cefazolin/rate of hydrolysis of cephaloridine versus rate of hydrolysis of benzylpenicillin/rate of hydrolysis of cephaloridine by whole-cell suspensions of S. aureus. Values for the reference isolates which produce the A (NCTC 9789), B (22260), C (3804), and D (FAR 10) serotypes of S. aureus β-lactamase are identified by squares. The values for the 22 β-lactamase-producing phage group II isolates excluding reference strain 22260 are represented by circles. Compared to the ratio for nitrocefin/cefazolin used in our 1990 report of whole-cell methods for the typing of staphylococcal β-lactamases (17), the use of a ratio for benzylpenicillin/cephaloridine enables easier discrimination between the type B versus type C enzyme and between the type A versus type D enzyme.

FIG. 2

Agarose gel (a) and Southern hybridization with a blaZ probe (b) of EcoRI-restricted plasmid DNA from phage group II, β-lactamase-producing isolates of S. aureus. Isolates include NCTC 9789, type A control (lane 1); RN9, type C control (lane 2); 3804, type C control (lane 3); FAR10, type D control (lane 4); 22260, type B control (lane 5); ST79/741 (lane 6); DK1133 (lane 7); DK5150 (lane 8); DK6024 (lane 9); DK1041 (lane 10); DK2027 (lane 11); DK3086 (lane 12); DK5110 (lane 13); DK5154 (lane 14); DK5162 (lane 15); and DK5246 (lane 16). Electrophoresis was performed on a single gel, the photograph of which was cut and reassembled for clarity of presentation. The type of β-lactamase produced by each isolate is noted above the lane number. The positions of base pair markers are indicated. NCTC 9789 has previously been shown to carry the type A β-lactamase gene on the chromosome rather than a plasmid (3).

FIG. 3

Southern hybridization of chromosomal DNA from phage group II, type B β-lactamase-producing isolates of S. aureus using a blaZ probe. Lanes 1 to 9, EcoRI-restricted DNA; lanes 10 to 18, HindIII-restricted DNA. Isolates include NCTC 9789 (lanes 1 and 10), 22260 (lanes 2 and 11), ST79/741 (lanes 3 and 12), DK1133 (lanes 4 and 13), DK2034 (lanes 5 and 14), DK5143 (lanes 6 and 15), DK5150 (lanes 7 and 16), DK6024 (lanes 8 and 17), and DK6026 (lanes 9 and 18). The positions of base pair markers are indicated. NCTC 9789 has previously been shown to carry the type A β-lactamase gene on the chromosome rather than a plasmid (3).

FIG. 4

Comparison of the nucleotide sequence of the gene encoding type B β-lactamase from strain 22260 to other S. aureus β-lactamases. The sequences for the type A, C, and D β-lactamases have been reported previously (4, 6, 10, 11, 38). Differences in nucleotide sequences are shown; −, no change from the blaZ sequence of the type A gene. Amino acid substitutions in the B, C, and D enzymes compared to the type A sequence are indicated above the codons. Amino acid numbering is according to Ambler and colleagues (1, 2), including gaps at residue 58 and residues 85 to 86 (2). L1 to L24 refer to the leader peptide. Whereas the sequences of the type A, C, and D β-lactamases have been determined through the carboxy-terminal residue at Ambler position 290, reliable sequence data for the type B enzyme were obtained through residue 253.