International Journal of Antimicrobial Agents 12 (1999) 333 – 339 Original article In vitro selection of glycopeptide-resistant variants of Enterococci Maria Puntorieri, Viviana Cafiso, Maria Santagati, Calogero Messina, Cinzia Azzarelli, Valentina Catalano, Giovanni Bonfiglio, Nicoletti Giuseppe, Stefani Stefania * Department of Microbiological and Gynaecological Sciences, Section of Microbiology, Uniersity of Catania, Via Androne 81, 95124 Catania, Italy Received 25 January 1999; accepted 8 April 1999 Abstract In order to study the possible phenotypic and genotypic changes related to glycopeptide pressure on enterococci, a study was undertaken using stepwise in vitro exposure to achieve the following objectives: (i) to evaluate the development of resistance and cross-resistance between vancomycin and teicoplanin; (ii) to determine the stability of the acquired level of resistance; (iii) to determine the phenotypic and genotypic changes related to glycopeptide pressure; and (iv) to assess the spectrum of antibiotic-sus- ceptibility of all strains. Our results showed that no variants resistant to glycopeptides could be selected after in vitro glycopeptide exposure experiments. However some strains showed increased MIC values: 8 mg/l to vancomycin in eight strains selected by vancomycin itself, while teicoplanin produced intermediate values to vancomycin in only three strains. The phenotypes were stable in vitro after numerous passages in antibiotic-free medium and three out of nine strains with a changed MIC level, showed 40, 42 and 43 kDa proteins in cell membrane preparations. The profile of antibiotic resistance was comparable in all isogenic strains tested with the exception of three selected strains that became susceptible to penicillin G. The pressure produced by glycopeptides, particularly vancomycin has contributed to an increased level of MIC that can influence the acquisition and/or full expression of this resistance. © 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Enterococci; Selection of resistance; Glycopeptides; Vancomycin; Membrane proteins www.elsevier.com/locate/isc 1. Introduction Among the most dramatic and clinically worrying examples of resistance to antimicrobials in recent years, has been the emergence and spread of vancomycin resistance in enterococci. In the USA, the incidence of nosocomial resistant strains to vancomycin reported to the National Nosocomial Infections Surveillance Sys- tem of the Centers for Disease Control and Prevention (NNIS), has increased from 0.3% in 1989 to 7.9% in 1993 [1] and now, up to 18% [2]. A survey performed in 1995, showed that the level of vancomycin resistance in Europe was 2% [3] and recent European data presented at the Eighth Annual Meeting of the Society of Health- care Epidemiology of America (Orlando, 1998) showed that VanA resistance in enterococci ranged from 0.2 to 3%; VanB are still rare and VanC ranged from 0.4 to 12% demonstrating that vancomycin resistance in Eu- rope is still not a clinical problem. Acquisition of glycopeptide resistance results from the transfer of a mobile genetic element that encodes enzymes for syn- thesis of low-affinity precursors and eliminates the high-affinity ones normally produced by the host. The sudden and widespread emergence of this prob- lem is particularly curious because vancomycin has been in clinical use since 1958. The long delay between the initial use of glycopeptides and the emergence of its resistance is rather surprising when compared with the situation of other antibiotic families. One of the logical explanations for the high levels of resistance is an increase in environmental vancomycin exposure. An escalated use of glycopeptides occurred during 1981 – * Corresponding author. Tel.: +39-95-311-352; fax: +39-95-325- 032. E-mail address: stefanis@mbox.unict.it (S. Stefania) 0924-8579/99/$ - see front matter © 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII:S0924-8579(99)00082-5