FEMS Microbiology Letters 119 (1994) 263-270 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier 263 FEMSLE 06000 Cloning and expression of the penicillinase from a borderline methicillin-susceptible Staphylococcus aureus strain in Escherichia coli Orietta Massidda *, Maria Pia Montanari, Marina Mingoia and Pietro Emanuele Varaldo lstituto di Microbiologia, Universith degli Studi di Ancona, Via Ranieri, Monte d'Ago, 60131 Ancona, Italy (Received 31 March 1994; accepted 4 April 1994) Abstract: The blaZ gene contained in a single 17.2-kb/3-1actamase plasmid from a borderline methicillin-susceptible Staphylococ- cus aureus strain (a53) has been cloned in Escherichia coli. A Bluescript II derivative in which the ampicillin resistance gene has been replaced with the chloramphenicol resistance gene was used as a multi-copy vector. One ampicillin-resistant colony was detected among 31 chloramphenicol-resistant transformants selected. This E. coli clone harbored a recombinant plasmid (pAHI2) containing two different staphylococcal HindllI inserts (7.0 and 5.3 kb), of which only the former hybridized with a blaZ probe. The clone showed an ampicillin MIC of > 1024 p.g ml- l, independently of the inoculum size used, and produced large amounts of /3-1actamase, which hydrolyzed nitrocefin and penicillin G but not methicillin of the /3-1actamase substrate, padac. In contrast, S. aureus a53 hydrolyzed all four substrates. The fact that high levels of staphylococcal penicillinase are unable to cause methicillin hydrolysis confirms that penicillinase hyperproduction is unlikely to be the true mechanism responsible for the borderline phenotype. These results also suggest that the two different /3-1actamases (penicillinase and methicillinase) associated with borderline S. aureus strains have a different genetic origin. Key words." /3-Lactamase; Staphylococcus aureus; Borderline susceptibility; Penicillinase-resistant penicillin Introduction Particular attention has recently been focused on the problem of Staphylococcus aureus strains with borderline levels of susceptibility or resis- tance to methicillin and related anti-staphylococ- cal penicillinase-resistant penicillins [1]. That bor- derline S. aureus strains are not intrinsically re- sistant or heteroresistant was first suggested by * Corresponding author. Tel. (071) 220 4697; Fax: (071) 220 4693. McDougal and Thornsberry [2] and then con- firmed by the experimental findings that these strains do not produce penicillin-binding protein (PBP) 2a [3] - i.e. the biochemical correlate and marker of intrinsic methicillin resistance - and that their DNA does not hybridize with probes specific for the methicillin resistance determinant [4]. Since borderline S. aureus strains generally produce large amounts of /3-1actamase and be- come fully susceptible to antistaphylococcal peni- cillins in the presence of /3-1actamase inhibitors, McDougal and Thornsberry originally hypothe- sized this borderline susceptibility or low-level SSDI 0378-1097(94)001 46-I Downloaded from https://academic.oup.com/femsle/article/119/3/263/544084 by guest on 02 March 2022