Among clinical isolates of Staphylococcus aureus tested for resistance to the antibiotic bleomycin, 197 were found to be resistant; most of them were also ...
Journal of Antimicrobial Chemotherapy (1996) 37, 65-75
Bleomycin resistance in Staphylococcus aureus clinical isolates Dimitra Gennimata*-*, Julian Davies*'* and Asterios S. Tsiftsoglou"t
"Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, Thessaloniki 546 06, Greece; bMicrobial Engineering Unit, Institut Pasteur, Paris, France
Introduction
Bleomycin is a potent antibiotic used in the therapy of various neoplasms (Carter, Crooke & Umezawa, 1978). Surprisingly, bacteria isolated from clinical samples, including those originating in cancer hospitals (which may be a possible ecological niche), often encode resistance to bleomycin. The genetic determinants for bleomycin resistance identified so far, have been found on transposon Tn5 in Gram-negative bacteria (Genilloud, Garrido & Moreno, 1984) and on plasmids related to pUBl 10 in staphylococci (Semon et al., 1987). Genes encoding bleomycin-binding proteins (BBP) have also been identified in streptomycetes producing antibiotics of the bleomycin class (Gatignol, Durand & Tiraby, 1988; Suguyama et al., 1994). The bleomycin resistance genes isolated from different sources exhibit no nucleotide sequence similarities, while the corresponding proteins display limited structural similarity (Gatignol, 1987; Semon et al., 1987). With the exception of Tn5, the mechanism of resistance to bleomycin has been shown to be due to stoichiometric binding of bleomycin to a bleomycin-binding protein that prevents DNA damage (Gatignol et al., 1988). However, a bleomycin acetyltransferase has also been identified in bleomycin producing organisms (Suguyama et al., 1994). *Present address: Department of Microbiology, University of British Columbia, 300-6174 University Boulevard, Vancouver, BC V6T 123, Canada. tAll correspondence should be addressed to Prof. A. S. Tsiftsoglou.
0305-7453/96/010065 + 11 $12.00/0
65 •£ 1996 The British Society for Antimicrobial Chemotherapy
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Among clinical isolates of Staphylococcus aureus tested for resistance to the antibiotic bleomycin, 197 were found to be resistant; most of them were also resistant to tobramycin and contained plasmids. DNA dot-blot hybridization analysis of the bleomycin resistant isolates with an 171 bp probe derived from plasmid pUBHO indicated that 43 strains (22%) carried pUBHO-like bleomycin resistance DNA sequences. Analysis of bacterial cell lysates derived from the bleomycin resistant isolates indicated that many contained bleomycin-binding proteins (BBP), that prevent DNA damage by the antibiotic. Of 13 strains that were analysed by DNA gel electrophoresis and Southern blot DNA hybridization, six were found to carry pUBl 10-like bleomycin resistance DNA sequences. These studies indicate that there may be more than one genetic determinant for bleomycin resistance in S. aureus whose DNA is protected.
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In an initial study (Semon et al., 1987), only a limited number of staphylococci were screened for resistance to bleomycin; the results suggested that bleomycin resistance is widespread and often associated with resistance to aminoglycosides in this genus. In the present study, 254 independent clinical isolates of Staphylococcus aureus were examined for resistance to bleomycin and other antibiotics. DNA dot-blot hybridization analysis has been applied extensively for the identification of antibiotic resistance genes and their dissemination in bacteria (Ounissi et al., 1990; Courvalin, 1991). Since the staphylococcal plasmid pUBHO encoding resistance to aminoglycosides had been shown to carry a bleomycin resistance determinant (Semon et al., 1987), we investigated whether resistance to bleomycin in S. aureus was mainly due to this gene, to other sequence-related genetic determinants or to an entirely different mechanism of resistance. Furthermore, we investigated whether the genetic determinants are located on the bacterial chromosome, on plasmids or on both.
Bacterial strains, plasmids and phages Gram-positive clinical isolates, used in this study were kindly provided by Drs Horaud and Nevine El Sohl of the Laboratory of Bacteriology at the Pasteur Institute, Paris. They were 254 independent isolates, representing S. aureus strains collected during a year (1988) in France. Among them were 18 methicillin sensitive S. aureus (MSSA), 13 methicillin resistant S. aureus (MRSA) and 23 gentamicin and methicillin resistant S. aureus (GMRSA) strains of phage type 77, that have been described previously for their serotype, phage type, antibiotic resistance patterns and typing using DNA probes (Monzon-Moreno et al., 1991). However, in our study, all 254 strains were used, regardless of special characteristics. Bacillus subtilis and S. aureus BM103 harbouring plasmid pUBHO were from the laboratory collection. Bacteria were tested for bleomycin resistance (BleR) by plating on to L-agar plates containing 20 mg/L bleomycin and for resistance to other antibiotics by the disc agar diffusion assay. The cloning vector M13mpl9 digested with the restriction enzyme AccI was purchased from Appligene, Illkirch, France. The plasmid pUBHO (McKenzie et al., 1987; Semon et al., 1987) was used as source for the excision of the bleomycin resistance gene probes as well as a positive control for hybridization studies.
Chemicals and biological reagents Restriction enzymes used throughout this study (£coRI, Hindlll, BamHl, Bglll, HpaU, MbolY) were purchased from BRL, Bethesda, MD, USA and used according to the manufacturer's instructions. Bleomycin was kindly donated by Nippon Kayaku, Japan. Discs containing ampicillin (10 fig), gentamicin (10 fig), tobramycin (10 ^g), amikacin (30 fig), kanamycin (20 fig), neomycin (20 fig) were from Diagnostics Pasteur, Mames-la-Coquette, France. Ampicillin, tetracycline, X-gal and IPTG (isopropyl -D-thiogalacto-pyranoside) were from Sigma Chem. Co., MD., USA. [
